Differential Stabilities and Sequence-Dependent Base Pair Opening Dynamics of Watson–Crick Base Pairs with 5-Hydroxymethylcytosine, 5-Formylcytosine, or 5-Carboxylcytosine (original) (raw)

Cytosine methylation by DNA methyltransferases (1-4) to form 5-methylcytosine (5mC) (5) is important in epigenetic regulation of the eukaryotic genome. (6, 7) The reconversion of 5mC to cytosine during active demethylation (8-25) involves the stepwise oxidation of 5mC. Oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) (10, 26, 27) is accomplished by ten-eleven translocation (TET) dioxygenases (15, 28-30) and occurs in response to oxidative stress as a consequence of UV radiation. (31) Further oxidation of 5hmC by TET dioxygenases forms 5-formylcytosine (5fC) (27) and 5-carboxylcytosine (5caC). (8, 22, 27-30, 32) These have been detected in cellular DNA. (32, 33) Both 5fC and 5caC, but not 5hmC, are substrates for thymine DNA glycosylase (TDG), (14) consistent with the greater abundance of 5hmC in mammalian tissues (9) and implicating catalyzed base excision of oxidized 5mC derivatives in active demethylation.

The differential processing of 5fC and 5caC vs 5hmC by TDG could be mediated by their differential recognition in DNA. Recently, Raiber et al. (34) reported that a DNA dodecamer containing three 5fC sites in an iterated CG repeat sequence exhibited 5fC-specific helical unwinding, due to specific changes in the geometry of the grooves and base pairs involving 5fC. DNA glycosylases may also exploit differential base pair opening rates as a basis for substrate recognition. For example, enhanced base pair opening rates at A:U base pairs facilitate the recognition of uracil by uracil DNA glycosylase (UDG). (35) It has also been hypothesized that TDG recognizes wobble base pairing geometry at oxidized cytosines, (17, 19, 23, 36, 37) as the imino tautomers of 5caC or 5fC may adopt wobble-like base pairs with the complementary G. (20, 37) However, calculations of the stabilities of the amino and imino tautomers of 5fC and 5caC at the nucleobase level have suggested that, when paired with G, both 5fC and 5caC, which are substrates for TDG, (14) preferentially form Watson–Crick pairs. (23) Alternatively, as proposed by Maiti et al., (14) the differential processing by TDG could be mediated by differences in the corresponding transition state catalytic complexes involving 5fC, 5caC, or 5hmC. Maiti et al. (14) proposed that the preferential excision of 5fC and 5caC by TDG is facilitated by the presence of electron-withdrawing substituents at the C5 carbon for these two oxidized cytosines. This electron-withdrawing effect (14, 23) would be anticipated to stabilize developing negative charge in the transition state complex for base excision.

Here, we have incorporated 5hmC, 5fC, or 5caC into the 5′-T8X9G10-3′ sequence of the self-complementary Dickerson–Drew dodecamer (DDD), (38, 39) which contains the 5′-CG-3′ sequence associated with genomic cytosine methylation, forming DDDhm, DDDf, and DDDca duplexes (Chart 1), respectively. Importantly, the DDD is amenable to crystallographic (38-43) and spectroscopic (44-47) analyses. The characterization of the DDDhm, DDDf, and DDDca duplexes by thermal melting studies, measurements of base pair opening dynamics, crystallography, and NMR reveals lesion- and sequence-specific differences among 5hmC, 5fC, or 5caC in the 5′-T8X9G10-3′ sequence, which may be relevant to their recognition by TDG. Relative to 5hmC and 5fC, incorporation of 5caC increases the stability of the DDD. This is reflected in reduced base pair opening dynamics for DDDca, as compared to that for DDDhm and DDDf, at neighboring base pair A5:T8. Similar, but smaller, differences in base pair opening dynamics are observed at the oxidized base pair G4:X9, whereas minimal effects are observed at neighboring base pair C3:G10. No evidence for wobble base pairing interactions involving the oxidized cytosines is observed; each of these oxidized cytosines favors Watson–Crick base pairing. These sequence-specific differences in the DDD may be related to the recognition of these oxidized cytosines by TDG. However, they differ from the sequence-specific effects observed by Raiber et al. (34) for an iterated CG repeat containing three 5fC sites. Moreover, they do not correlate with the differential ability of TDG to excise 5fC and 5caC vs 5hmC, (14) which may be mediated by differences in the transition state complex for base excision.

Chart 1

Chart 1. (A) Structures of C, 5mC, 5hmC, 5fC, and 5caC and (B) Sequences and Numbering of the Nucleotides for DDD and Oxidized DDD Duplexesa

Chart aIn solution, the two strands of the DDD exhibit pseudo-dyad symmetry. NMR resonances of symmetry-related nucleotides in the two strands are not individually observed. In the crystal, corresponding nucleotides from paired strands are not symmetry-related, and nucleotides are numbered individually. DDDm, DDDhm, DDDf, and DDDca refer to the DDD containing 5mC, 5hmC, 5fC, and 5caC, respectively.

Experimental Procedures

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Oligodeoxynucleotide Synthesis

Oligodeoxynucleotides were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified by anion-exchange HPLC. The DDDhm duplex was prepared with an Expedite 8909 DNA synthesizer (PerSeptive Biosystems) on a 1 μmol scale using ethylcyanide-protected 5-hydroxymethyl-dC, phenoxyacetyl-protected dA, 4-isopropyl-phenoxyacetyl-protected dG, acetyl-protected dC, and dT phosphoramidites and solid supports (Glen Research, Inc., Sterling, VA). The modified phosphoramidite was incorporated by removing the column from the synthesizer and sealing it with two syringes, one of which contained 250–300 μL of the manufacturer’s 1H-tetrazole activator solution (1.9–4.0% in CH3CN, v/v) and the other contained 250 μL of the modified phosphoramidite solution (15 mg in anhydrous CH3CN). The 1H-tetrazole and the phosphoramidite solutions were sequentially drawn through the column (1H-tetrazole first), and this procedure was repeated over 30 min. The column was washed with anhydrous CH3CN and returned to the synthesizer for capping, oxidation, and detritylation steps. The deprotection was accomplished with 30% NH4OH for 17 h at 75 °C.

Oligodeoxynucleotide Purification and Characterization

Oligodeoxynucleotides were purified by semipreparative HPLC at 260 nm (Atlantis, Waters Corporation, C18, 5 μm, 250 mm × 10.0 mm). The column was equilibrated either with 30 mM sodium phosphate (pH 7.0) (for DDDm, DDDhm, DDDca) or 0.1 M ammonium formate (pH 6.5) (for DDDf). The gradient was 1–15% CH3CN over 20 min, 15–80% CH3CN over 5 min, and 1% CH3CN over 5 min, at 4.5 mL/min. Oligodeoxynucleotides were desalted by passing over G-25 Sephadex (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Oligodeoxynucleotides were characterized by MALDI-TOF mass spectrometry (calcd for DDD [M – H]−m/z 3646.4, found m/z 3647.8; calcd for DDDm [M – H]−m/z 3660.5, found 3663.4; calcd for DDDhm [M – H]−m/z 3675.5, found 3679.7; calcd for DDDf [M – H]−m/z 3674.4, found 3673.2; calcd for DDDca [M – H]−m/z 3690.4, found 3693.1). Oligodeoxynucleotides were prepared in 100 mM NaCl, 50 μM Na2EDTA, in 10 mM sodium phosphate (pH 7.0), heated at 85 °C for 15 min, and annealed by cooling to room temperature. Duplex concentrations were determined by UV absorbance, using extinction coefficients calculated at 260 nm. (48)

Thermal Denaturation

The concentration of DNA was 1.2 μM. Measurements were conducted in 100 mM NaCl, 50 μM Na2EDTA, in 10 mM sodium phosphate (pH 7.0). The temperature was increased from 10 to 80 °C at 1 °C/min. _T_m values were calculated from first-order derivatives of 260 nm absorbance vs temperature profiles. (49)

NMR

Spectra were obtained at 900 MHz using a 5 mm cryogenic probe (Bruker Biospin Inc., Billerica, MA). Oligodeoxynucleotides were prepared at a duplex concentration of 0.25 mM in 180 μL of 100 mM NaCl, 50 μM Na2EDTA, 11 mM NaN3, in 10 mM sodium phosphate (pH 7.0). The samples were exchanged with D2O and dissolved in 180 μL of 99.996% D2O to observe nonexchangeable protons. NOESY (50) spectra were collected in 99.996% D2O to observe nonexchangeable protons. The temperature was 15 °C. TPPI quadrature detection was used, and data were collected at a mixing time of 250 ms. The relaxation delay was 2.0 s. Data were recorded with 2k real points in the t2 dimension and 1k real points in the t1 dimension. Spectra were zero-filled during processing to create a 2k × 2k matrix. Chemical shifts were referenced to the chemical shift of water at the corresponding temperature, with respect to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). To observe exchangeable protons, samples were prepared in 9:1 H2O/D2O. For observation of imino protons, spectra were recorded at 5, 15, 25, 35, 45, and 55 °C. NOESY spectra were collected at 5 °C with 70 or 250 ms mixing times and relaxation delay of 2.0 s. Water suppression was achieved by the Watergate pulse sequence. (51) Data were processed with TOPSPIN (2.0.b.6, Bruker Biospin Inc., Billerica, MA).

Base Pair Opening

NMR data were collected at 500 MHz using a 5 mm cryogenic probe, at 15 °C. Samples were in 180 μL of 9:1 H2O/D2O containing 100 mM NaCl, 50 μM Na2EDTA, 11 mM NaN3, 1 mM triethanolamine, in 10 mM sodium phosphate (pH 8.9). (52-56) The presence of triethanolamine enabled the pH of the sample to be monitored during the titration, in situ, by measuring the chemical shift difference between the two methylene groups. (52) Magnetization transfer from water to the imino protons was followed by observation of the imino proton resonances after variable mixing times. (57, 58) Selective spin inversion of the water protons was achieved with a 2 ms 180° sinc pulse with 1000 points. To minimize effects of radiation damping during the mixing time, a 0.1 G cm–1 gradient was used. Water suppression was achieved by a binominal 1–1 echo sequence, jump and return, (59) with flanking 1 ms smooth square shape gradients, 15 G cm–1. Sixteen values of the delay ranging form 1 ms to 15 s were used. Data were processed in TOPSPIN. Ammonia, p_K_a of 9.2 at 15 °C, was the proton acceptor. (57) Data analyses were performed using PRISM (v. 6.0b, GraphPad Software, Inc., La Jolla, CA). Exchange rates were calculated using established methods. (60, 61) In order to determine rates of base pair opening, exchange rates were plotted against concentrations of the active form of the ammonia base catalyst. Equilibrium constants for base pair opening were calculated by fitting exchange rate data as a function of ammonia concentration. (52)

Crystallization and X-ray Diffraction

Crystals were grown at 18 °C over 8 to 16 days by hanging-drop vapor diffusion, using the nucleic acid mini-screen (Hampton Research, Aliso Viejo, CA). Droplets of 2 μL containing 1.2 mM duplex in precipitant solution were equilibrated against 0.75 μL of 35% MPD. The solution compositions are summarized in Table S1 in the Supporting Information. Single crystals were mounted in nylon loops and flash-frozen in liquid nitrogen.

For DDDhm, data were collected on the 19-ID beamline of the Structural Biology Center at the Advanced Photon Source (APS) of Argonne National Laboratory (ANL, Argonne, IL). (62) The wavelength was 0.9794 Å. Initial indexing and scaling of diffraction images and further reflection merging was done using HKL3000. (63) To ensure completeness of the data, two passes were collected. For DDDca, data were collected on the 24-IDC beamline of the Northeastern Collaborative Access Team (NE-CAT) at the APS (ANL). The wavelength was 0.97920 Å. Initial indexing and scaling of diffraction images, together with reflection merging, were done using XDS (64, 65) and SCALA (66) in the CCP4 (67) suite as part of the RAPD data collection strategy at NE-CAT. For DDDf, data were collected on the 21-IDD beamline of the Life-Sciences Collaborative Access Team (LS-CAT) at the APS (ANL). The wavelength was 1.000 Å. Initial indexing and scaling of diffraction images, together with reflection merging were done in HKL2000. (68) Details are shown in Table 1.

Table 1. Crystal Data, Data Collection, and Refinement Statistics for the DDDhm, DDDf, and DDDca Duplexes

Crystal Structure Determination and Refinement

Structures were determined by molecular replacement using the DDD as the search model (PDB ID code 436D). (40) Molecular replacement searches were completed with MOLREP (69, 70) in the CCP4 suite. (67) An initial model was checked and rebuilt in COOT. (71) The model was rebuilt and further refined using REFMAC. (72, 73) Final models were refined against all reflections, except for 5% randomly selected reflections used for monitoring _R_free. The refinement statistics are presented in Table 1.

Data Deposition

Complete structure factors and data coordinates were deposited in the Protein Data Bank (http://pdb.org): PDB ID code 4I9V for DDDhm, 4QC7 for DDDf, and 4PWM for DDDca.

Results

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Stabilities of the Duplexes Containing Oxidized Cytosines

The impact of placing 5hmC, 5fC, or 5caC site specifically into the 5′-CG-3′ sequence was investigated by incorporating each oxidized cytosine into the 5′-T8X9G10-3′ sequence of the DDD. (38, 39) The _T_m values of the duplexes were obtained in 100 mM NaCl at pH 7. They were compared to both the unmodified DDD and also to the DDD containing 5mC in the 5′-T8X9G10-3′ sequence (DDDm). The _T_m of the DDD duplex was 48 °C, the _T_m of the DDDm duplex was 46 °C, the _T_m of the DDDhm duplex was 48 °C, and the _T_m of the DDDf duplex was 46 °C. These small differences in _T_m suggested that the presence of 5mC or of the oxidized cytosines 5hmC or 5fC in the 5′-T8X9G10-3′ sequence did not greatly affect the _T_m of the DDD. In contrast, the _T_m of the DDDca duplex increased to 54 °C. NMR spectra of the exchangeable guanine N1H and thymine N3H imino protons were recorded from 5–55 °C (Figure 1). The resonances were assigned using standard methods. (74) For the DDDca duplex, the G4 N1H proton remained sharp at 55 °C, consistent with the increased _T_m value associated with the 5caC nucleobase in the 5′-T8X9G10-3′ sequence. At the neighbor A5:T8 base pair, the T8 N3H resonance remained detectable at 55 °C, although it exhibited broadening. At the neighbor C3:G10 base pair, the G3 N1H resonance remained detectable at 55 °C, also exhibiting broadening. The stabilizing effect extended two base pairs in each direction, also including the imino protons of base pairs G2:C11 and A6:T7. In contrast, for the DDDhm duplex at the oxidized G4:X9 base pair, the G4 N1H resonance was severely broadened at 55 °C. Likewise, the corresponding resonance in the DDDf duplex was severely broadened at 55 °C. At the neighboring base pair C3:G10, the G10 N1H resonance in the DDDf duplex broadened at 35 °C. The T8 N3H resonances broadened at 45 °C in the DDDf duplex and at 55 °C in the DDDhm duplex. The temperature dependence of line widths of the imino resonances is shown in Figure S1 of the Supporting Information.

Figure 1

Figure 1. 1H NMR of imino proton resonances as a function of temperature for (A) DDD, (B) DDDm, (C) DDDhm, (D) DDDf, and (E) DDDca. Data were collected at 900 MHz.

Base Pair Opening Dynamics

Magnetization transfer from water after variable times was followed by observation of the guanine N1H and thymine N3H resonances, at 15 °C. The imino proton exchange rates were measured in the absence and the presence of added ammonia base catalyst. (45, 52, 57, 58, 61, 75) The exchange with water follows a two-state model, where the base pair undergoes a conformational change from the closed to the open state, from which proton exchange occurs. (57, 75) The open base pair is exchange-competent because the proton is accessible to acceptors in solution. As described by Russu and co-workers, (75-77) in the EX1 regime, the concentration of acceptors is sufficient for rapid exchange from the open state (_k_ex,open ≫ _k_cl), so exchange occurs at each opening event and _k_ex = _k_op. In the EX2 regime, where the concentration of base is low (_k_ex,open ≪ _k_cl), the rate of exchange from the open state is proportional to the exchange rate and the concentration of the acceptor. (75-77)

Figure 2 shows the results for the C3:G10, G4:X9, A5:T8, and A6:T7 base pairs of the DDDhm, DDDf, and DDDca duplexes. They were compared to both the unmodified DDD and to the DDDm duplexes. Plots of exchange rates as a function of ammonia concentration suggested that the EX1 regime (75-77) was attained. Consistent with the results of Moe et al., (45) the rates of imino proton exchange were lower for G:C base pairs C3:G10 and G4:X9 and greater for A:T base pairs A5:T8 and A6:T7. At 15 °C, the oxidized cytosines differentially altered exchange rates of the imino protons of the C3:G10, G4:X9, A5:T8, and A6:T7 base pairs. The greatest effects were observed at the neighbor A5:T8 base pair. For the DDDhm and DDDf duplexes, the exchange rate of the A5:T8 base pair imino proton increased at all concentrations of ammonia (Figure 2). There was a 3-fold increased rate of base pair opening in the DDDhm duplex and a 5-fold increased rate of base pair opening in the DDDf duplex, with respect to the DDD duplex (Table 2). In contrast, for the DDDca duplex, the exchange rate of the A5:T8 imino proton was similar to those of the DDD and DDDm duplexes at all concentrations of ammonia. These differences were reflected in measurements of the respective equilibrium constants for base pair opening. For the DDDhm duplex, the equilibrium constant for base pair opening (α_K_op) at base pair A5:T8 was 7.3 × 108, and for the DDDf duplex, the equilibrium constant for base pair opening at A5:T8 was 1.1 × 109, differing from the DDD and DDDm duplexes (3.4 × 108 and 3.5 × 108, respectively). In contrast, for the DDDca duplex, the equilibrium constant for base pair opening of A5:T8 was 4.1 × 108, similar to that of the DDD and DDDm duplexes. The neighbor effect did not extend beyond the A5:T8 base pair. At base pair A6:T7, exchange rates as a function of ammonia concentration were comparable for all duplexes.

Figure 2

Figure 2. Plots showing imino proton exchange rates obtained by monitoring magnetization from water as a function of ammonia base catalyst: (A) base pair C3:G10, (B) base pair G4:X9, (C) base pair A5:T8, and (D) base pair A6:T7 in the DDD (black), DDDm (green), DDDhm (blue), DDDf (red), and DDDca (pink) duplexes.

Table 2. Rate and Equilibrium Constants for DNA Base Pair Opening

A smaller effect on base pair opening dynamics was observed at the G4:X9 base pair. For the DDDf duplex, the exchange rate of G4:X9 was greater at all concentrations of ammonia than that for the DDDhm, DDDca, DDDm, and DDD duplexes (_k_op = 26 s–1 in DDDf vs _k_op = 16, 4, 7, and 8 s–1 for DDDhm, DDDca, DDDm, and DDD, respectively). For the DDDf duplex, the equilibrium constant for base pair opening increased 3-fold, calculated as 2.8 × 107 vs 1.2 × 107, 7.5 × 106, 1.2 × 107, and 6.0 × 106, respectively, for the DDD, DDDm, DDDhm, and DDDca duplexes.

In contrast, base pair opening dynamics at the neighboring C3:G10 base pair were not affected by the presence of the oxidized cytosines in the DDDhm, DDDf, or DDDca duplex. Thus, the differences in base pair opening dynamics for the 5hmC, 5fC, and 5caC bases in the 5′-T8X9G10-3′ sequence of the DDD exhibit a pronounced sequence dependence, with the greatest effects being evident at the neighboring A5:T8 base pair. This is the base pair located in the 5′-direction with respect to the oxidized cytosine X9. The overall results are summarized in Table 2.

Structures of the DDDhm, DDDf, and DDDca Duplexes

The modified DDDhm, DDDf, and DDDca duplexes yielded diffraction-quality crystals. Crystals belonged to the orthorhombic _P_212121 space group. The crystal structures were determined using the unmodified DDD (PDB ID code 436D) (40) as a search model for molecular replacement. Structures were refined using anisotropic B factors to a resolution of 1.02 Å for DDDhm and isotropic B factors to resolutions of 1.90 and 1.95 Å for DDDf and DDDca, respectively. Each of the structures was compared to that of the DDD. (40) Overall, the structures were similar to the DDD, (40) as indicated by comparative rmsd analyses, with rmsd values of 0.67, 0.46, and 0.49 Å for DDDhm, DDDf, and DDDca, respectively. Classical features of the DDD, including waters forming the minor groove spine of hydration, (78) were conserved. The data and refinement statistics are provided in Table 1.

Figure 3 shows electron density and base pairing arrangements for the 5hmC:G, 5fC:G, and 5caC:G base pairs in the DDDhm, DDDf, and DDDca duplexes, respectively. Watson–Crick base pairing was evident, and the hydroxymethyl, formyl, or carboxyl moieties of the oxidized cytosines were oriented into the major groove. The formyl group of 5fC and the carboxyl group of 5caC were within hydrogen-bonding range of the _N_4 exocyclic amines of the oxidized cytosines. For the DDDhm duplex, electron density associated with the hydroxymethyl moiety of 5hmC suggested partial occupancy of two conformations. The major conformation refined with occupancy 0.8, and the minor conformation refined with occupancy 0.2. In the major conformation, the hydroxyl group hydrogen bonded with the terminal N1 ammonium moiety of a spermine and with G10_O_6 via an ordered water molecule (Figure S2 of the Supporting Information). In the minor conformation, the hydroxyl group was oriented toward the backbone phosphate and formed interactions with neighboring waters (Figure S3 of the Supporting Information). A hydrogen bond was also observed between the hydroxyl group at the modified cytosine X21 and an axially coordinated water (HOH 12) at a distance of 2.7 Å, with a further interaction to G22 N7 (2.8 Å). An additional hydrogen bond was observed between the X21 hydroxyl and G22_O_6 via water HOH 11 (3.0 Å distance from X21 to HOH 11 and 2.7 Å from HOH 11 to G22_O_6) (Figures S2 and S3 of the Supporting Information). Base stacking patterns for the DDDhm, DDDf, and DDDca duplexes were similar (Figure 4).

Figure 3

Figure 3. Fourier (2_F_o – _F_c) sum electron density contoured at the 1.0σ level (green meshwork) around the (A) 5hmC:G, (B) 5fC:G, and (C) 5caC:G base pairs showing Watson–Crick base pairing geometry.

Figure 4

Figure 4. (A) DDDhm, (B) DDDf, and (C) DDDca structures, illustrating stacking interactions at oxidation sites.

The DDDhm, DDDf, and DDDca duplexes were also examined by NMR, (44, 46, 47) using standard methods. (79, 80) The sequential base aromatic → deoxyribose anomeric NOEs were identified from C1 → G12 (Figure S4 of the Supporting Information). For the DDDhm, DDDf, and DDDca duplexes (and as well for the DDD and DDDm duplexes), the intensities of NOE cross-peaks between the purine H8 and pyrimidine H6 protons and the deoxyribose H1′ protons were of the same relative magnitudes as those between other bases in the sequence, indicating that the glycosyl bonds maintained the anti conformations. In all instances, the NOE connectivity of the purine N1H and pyrimidine N3H protons (74) was obtained from G2:C11 → C3:G10 → G4:X9 → A5:T8 → A6:T7 (Figure 5). NOE cross-peaks from the oxidized base X9_N_4H1 and _N_4H2 protons to the complementary base G4 N1H proton were observed, as well as interactions to neighbor bases T8 N3H and G10 N1H, consistent with Watson–Crick geometry being favored, corroborating the crystallographic data (Figure 3). Significantly, evidence for intranucleotide hydrogen bonding involving the formyl group of 5fC or the carboxyl group of 5caC and the _N_4 exocyclic amine of 5fC or 5caC was evident in NMR spectra of the DDDf and DDDca duplexes, for which both of the X9_N_4 amino proton resonances shift downfield into the 7.8–8.8 ppm spectral range (Figure 5). The effect was most pronounced for the DDDca duplex. In contrast, for the DDD, DDDm, and DDDhm duplexes, one of the _N_4 amino protons shifts downfield, consistent with the maintenance of a Watson–Crick base pair, whereas the other remains in the 6.5–7.0 ppm spectral range, which is the anticipated result given that cytosine, 5mC, and 5hmC cannot form this hydrogen bond. Overall, the NMR data corroborated the crystallographic data, giving no indication of the presence of imino tautomers and suggesting that each of the oxidized cytosines participated in normal Watson–Crick base pairing when placed opposite guanine.

Figure 5

Figure 5. NOESY spectra depicting resonances for the thymine and guanine imino protons and sequential NOE connectivity for the imino protons of the base pairs G2:C11 to A6:T7 for (A) DDD, (B) DDDm, (C) DDDhm, (D) DDDf, and (E) DDDca duplexes (lower panels). Expansion of the NOESY spectra for (A) DDD, (B) DDDm, (C) DDDhm, (D) DDDf, and (E) DDDca duplexes (upper panels), illustrating the conservation of Watson–Crick base pairing and base stacking at the modification sites: a, C9 or X9_N_4H1 → T8 N3H; b, C9 or X9_N_4H2 → T8 N3H; c, C9 or X9_N_4H1 → G10 N1H; d, C9 or X9_N_4H2 → G10 N1H; e, C9 or X9_N_4H1 → G4 N1H; f, A5 H2 → G4 N1H; and g, C9 or X9_N_4H2 → G4 N1H. (Indices m, hm, f, or ca refer to the base pairs in the modified duplexes, DDDm, DDDhm, DDDf, and DDDca, respectively.) Data were collected at 900 MHz.

Discussion

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The 5hmC, (10, 26, 27) 5fC, (27) and 5caC (27, 29, 30) oxidation products of 5mC are intermediates in active demethylation (8-25) and have potential roles in epigenetic regulation of cellular function. (6, 7) Their removal is orchestrated by glycosylase-mediated base excision repair; TDG is essential for active DNA demethylation. (8) It has been reported that 5fC and 5caC, but not 5hmC, are substrates for thymine DNA glycosylase (TDG). (14) Accordingly, it was of interest to determine whether these oxidized cytosines differentially alter duplex DNA and how such differences correlate with differences in excision of 5hmC, 5fC, and 5caC by TDG. (14) The Dickerson–Drew dodecamer (DDD) (38, 39) provided a platform for conducting these studies. It contains the 5′-CG-3′ sequence associated with genomic cytosine methylation, and, most importantly, it is simultaneously amenable to crystallographic (38-43) and spectroscopic (44-47) analyses.

Stabilization of the DDD by 5caC

The presence of 5caC in the 5′-T8X9G10-3′ sequence stabilizes the DDD, as evidenced by the 6–8 °C increase in _T_m for the DDDca as compared to the _T_m values of the DDD and of the DDDm under the same conditions. NMR data for the base paired guanine N1H and thymine N3H imino protons (Figure 1) confirm this conclusion. For the DDDca duplex, at temperatures as high as 55 °C, the imino proton resonances of base pairs C3:G10, G4:X9, and A5:T8 remain detectable (Figure 1E). In contrast, for the DDDhm duplex and DDDf duplexes, the imino proton resonances of base pairs C3:G10, G4:X9, and A5:T8 broaden at temperatures above 35° (Figure 1C,D). While the inclusion of 5caC into the 5′-T8X9G10-3′ sequence in the DDD provides only a single data point for thermodynamic comparison, the observation that 5caC stabilizes the DDDca is consistent with calculations performed by Sumino et al. (81) It also corroborates data obtained by the same group for the stabilities of 13-mers and 14-mers containing 5caC. This stabilization of the DDDca is not attributable to improved base stacking geometry of 5caC in DDDca because 5caC exhibits a base stacking geometry in the DDD that is similar to both 5hmC and 5fC (Figure 4). However, electronic dipole–dipole interactions associated with 5caC (14, 23) might enhance the thermodynamic stability of the DDDca duplex without disturbing the stacking geometry.

Sequence-Specific Base Pair Opening Dynamics of the Oxidized Duplexes

The imino proton exchange rates at base pairs C3:G10, G4:X9, and A5:T8 depend upon the identity of the cytosine oxidation product and exhibit sequence dependence. The greatest effects are observed for the neighbor base pair A5:T8, with a smaller effect at G4:X9 and minimal effect at the neighbor base pair C3:G10. Base pair A5:T8 is the 5′-neighbor with respect to the oxidized cytosine at position X9, whereas base pair C3:G10 is the 3′-neighbor with respect to the oxidized cytosine at position X9 (Chart 1). While the A5:T8 base pair of the DDD intrinsically exhibits enhanced exchange kinetics, (45) the presence of either 5fC or 5hmC further enhances imino proton exchange rates at A5:T8, whereas the presence of 5caC does not (Figure 2), an observation that is consistent with the thermal stabilization of the duplex by 5caC as opposed to 5fC or 5hmC. Thus, for DDDf, base pair A5:T8 base pair opens with the frequency of _k_op = 222 s–1, five times faster than in the DDD. For the DDDhm duplex, base pair A5:T8 opens three times faster than in the DDD (_k_op = 110 s–1 vs _k_op = 40 s–1 in DDDhm and DDD, respectively).

Structures of Duplexes Containing 5hmC, 5fC, or 5caC

Evidence for wobble base pairing geometry at oxidized cytosines, (17, 19, 23, 36, 37) arising from imino tautomers of 5caC or 5fC, (20, 37) is not observed. The results are consistent with calculations of the stabilities of the amino and imino tautomers of 5fC and 5caC at the nucleobase level, which have suggested that, when paired with G, both 5fC and 5caC preferentially form Watson–Crick pairs. (23) Instead, each of the 5hmC, 5fC, and 5caC oxidation products favors Watson–Crick hydrogen-bonding interactions when located in the 5′-T8X9G10-3′ sequence (Figures 3–5).

A common structural feature of 5fC and 5caC in the DDD is formation of intranucleobase hydrogen bonds between the carbonyl oxygens of the formyl or carboxyl groups, respectively, and a cytosine _N_4H amino proton. This hydrogen bond had been observed between the exocyclic _N_4 amino group and the formyl oxygen at C5 of 5fC at the nucleoside level. (27, 82) The downfield shifts of both of the X9_N_4 amino proton resonances into the 7.8–8.8 ppm spectral range is evident in NMR spectra of the DDDf and DDDca duplexes (Figure 5) and is consistent with the formation of these hydrogen bonds. The NMR data corroborates the crystallographic structure data (Figure 3), which shows that the carbonyl oxygens of the formyl or carboxyl groups of 5fC or 5caC, respectively, and a cytosine _N_4H amino proton are within hydrogen-bonding distance. The effect in the NMR data is most pronounced for the DDDca duplex (Figure 5). In the crystallographic structure of the DDDca duplex (Figure 3), this hydrogen bond keeps the carboxyl group in plane with the oxidized cytosine. For the DDD, DDDm, and DDDhm duplexes, one of the _N_4 amino protons shifts downfield, consistent with the maintenance of a Watson–Crick base pair, whereas the other remains in the 6.5–7.0 ppm spectral range, which is the anticipated result given that cytosine, 5mC, and 5hmC cannot form this hydrogen bond.

Structure–Activity Relationships

DNA glycosylases (36) typically employ an extrahelical base-flipping mechanism (35, 83-89) to position substrates for catalysis. Differences in the ability of TDG to excise 5fC, 5caC, or 5hmC from DNA (14) could be mediated by differential recognition of these oxidized cytosine bases in DNA. Stivers et al. (61, 90-93) demonstrated that damage recognition by a different glycosylase, uracil DNA glycosylase (UDG), is facilitated by enhanced base pair opening rates for destabilized A:U base pairs. (35) The present data reveal that site- and sequence-specific differences with regard to duplex stability and base pair opening dynamics are observed when the 5hmC, 5fC, and 5caC are placed into the DDDhm, DDDf, and DDDca dodecamers within the 5′-T8X9G10-3′ sequence. Neither the stabilization of the DDD by 5caC nor the differences in base pair opening dynamics correlate with differences in the excision of 5hmC, 5fC, and 5caC by TDG, as reported by Maiti et al. (14) Both 5hmC and 5fC exhibit increased base pair opening rates at the neighboring A5:T8 base pair. However, only 5fC is excised by TDG. Moreover, 5caC, which also is excised by TDG, thermally stabilizes the DDDca and does not exhibit increased base pair opening kinetics at the 5′-neighbor A5:T8 base pair (Figure 2).

It has been proposed that the imino tautomers of 5caC or 5fC adopt wobble-like base pairing geometry with the complementary G, which might provide a basis for recognition by TDG. (20, 37) The present crystallographic and NMR data indicate that the 5hmC, 5fC, and 5caC bases each favor Watson–Crick base pairing in the DDD duplex. This argues against wobble base pairing involving imino tautomers of these oxidized cytosines as a primary mode of recognition by TDG. However, the presence of small amounts of the imino tautomers cannot be ruled out, nor can a shift from Watson–Crick base pairing to wobble pairing subsequent to enzyme binding. It has been proposed that the hydrogen bond between the exocyclic _N_4 amine and the formyl or carboxyl oxygen at C5 of the 5fC or the 5caC base might shift the equilibrium toward the imino tautomer (94, 95) and lead to protonation at N3 of the oxidized cytosine. (37, 96) Additionally, other factors such as electrostatic and steric contributions, which remain to be examined, might modulate the differential recognition of these oxidized cytosines by TDG.

Alternatively, differences in the ability of TDG to excise 5fC, 5caC, or 5hmC from DNA could be controlled by differences in the catalytic step of base excision, once the oxidized cytosine bases have been inserted into the active site of the glycosylase. Maiti et al. (97) implied a role of the conserved Asn140 in the chemical step and of the conserved Arg275 in nucleotide flipping into the active site. In additional studies, Maiti et al. (14) accounted for the differential excision ability of TDG with respect to 5hmC, 5fC, and 5caC by arguing that activity is greatest for oxidized cytosines possessing electron-withdrawing substituents at the C5 carbon, which stabilize developing negative charge in the transition state complex for base excision. Following their argument, 5fC is a good substrate and 5hmC is not. (14) At neutral pH, 5caC exists as an anion with p_K_a values of 2.4 for the carboxyl and 4.3 for the N3 position, (23) and catalysis is facilitated because the ionized carboxyl group lowers the p_K_a of cytosine and stabilization of the carboxyl by the exocyclic amine of cytosine creates an electron-withdrawing effect. (14, 23) Maiti et al. (23) demonstrated that the excision ability of TDG with respect to 5fC is pH-independent but that the excision of 5caC is acid-catalyzed. Moreover, Zhang et al. (25) found that TDG binds to 5caC with greater affinity than to 5fC, U, or T and proposed that residues Asn157, His151, and Tyr152 are involved in hydrogen bonds with the 5caC carboxyl group. Finally, the structure of TDG in complex with DNA containing a G:5hmU mismatch showed that TDG engages in hydrogen-bonding interactions with both 5hmU and 5caC. (19)

The present results are consistent with the proposal by Maiti et al., (14) in which the excision specificity of TDG for 5fC and 5caC vs 5hmC is dictated by differences in the enzyme–substrate complex transition state. Both 5fC and 5caC form hydrogen bonds between the carbonyl oxygens of their formyl or carboxyl groups, respectively, and a cytosine exocyclic _N_4H amino proton. The electron-withdrawing effect of the 5fC and 5caC substitutents (14, 23) should be enhanced by hydrogen bonding between the carbonyl oxygens of their formyl or carboxyl groups, respectively, and a cytosine exocyclic _N_4H amino proton. This would be anticipated to stabilize developing negative charge in the transition state complex for base excision.

Summary

The cytosine oxidation products 5hmC, 5fC, and 5caC exhibit differences in thermodynamics and base pair opening dynamics when placed into the 5′-T8X9G10-3′ sequence of the DDD, but these do not correlate with differences in the ability of TDG to excise these cytosine oxidation products. (14) While TDG may exploit thermodynamic and base pair opening dynamics in the recognition of oxidized cytosines in DNA, differences in the transition state complexes for the base excision step may be rate-limiting with respect to the chemical step of base excision. Of course, the 5′-T8X9G10-3′ sequence is just one sequence, and it will be of interest to further examine the sequence dependence of these effects, particularly in light of the recent report from Raiber et al. (34) showing that the presence of three 5fC sites in an iterated CG repeat sequence changes the geometry of the DNA grooves and base pairs containing the 5fC oxidation product. DNA glycosylases may exploit different mechanistic pathways toward base excision. The recognition of uracil by uracil DNA glycosylase (UDG) is reported to be facilitated by enhanced base pair opening rates at A:U base pairs. (35) Interestingly, the 5mC DNA glycosylase DEMETER (DME) removes 5mC, 5hmC, and 5caC but has no activity for 5fC. (99, 98) Its inactivity toward 5fC also does not seem to be correlated with 5fC base pair opening rates, and it does not seem to correlate with the electron-withdrawing effect of the 5fC and 5caC substitutents. (14, 23)

Supporting Information

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Tables S1: Crystallization conditions for the DDDhm, DDDf, and DDDca duplexes. Figure S1: Temperature dependence of line widths of the imino proton resonances of the DDD, DDDm, DDDhm, DDDf, and DDDca duplexes. Figure S2: Modification site of the DDDhm duplex displaying interactions between the modified 5hmC and 3′-flanking G22 through water molecules. Figure S3: Modification site of the DDDhm duplex displaying interactions between the modified 5hmC and 3′-flanking G22 through water molecules. Figure S4: Expanded plots from NOESY spectra, depicting sequential NOE connectivities of the DDD, DDDm, DDDhm, DDDf, and DDDca duplexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Acknowledgment

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We thank Edward Hawkins (deceased), Dr. Plamen Christov, and Professor Carmelo J. Rizzo for help and guidance with synthesis of oligodeoxynucleotides.

Abbreviations

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5caC 5-carboxylcytosine
5fC 5-formylcytosine
5hmC 5-hydroxymethylcytosine
5mC 5-methylcytosine
DDD Dickerson–Drew dodecamer
DME DNA glycosylase DEMETER
DSS 4,4-dimethyl-4-silapentane-1-sulfonic acid
EDTA ethylenediaminetetraacetic acid, sodium salt
MPD 2-methyl-2,4-pentanediol
NOE nuclear Overhauser effect
NOESY two-dimensional nuclear Overhauser enhancement spectroscopy
TDG thymine DNA glycosylase
TET ten-eleven translocation dioxygenase
UDG uracil DNA glycosylase

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    The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair (BER) pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, resp., the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addn., TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA contg. a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1' hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.
  20. 20
    Hashimoto, H., Zhang, X., and Cheng, X. (2012) Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation Nucleic Acids Res. 40, 8276– 8284
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    Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
    Hashimoto, Hideharu; Zhang, Xing; Cheng, Xiaodong
    Nucleic Acids Research (2012),40 (17),8276-8284CODEN:NARHAD; ISSN:0305-1048. (Oxford University Press)
    The mammalian DNA glycosylase - methyl-CpG binding domain protein 4 (MBD4) - is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, resp., the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA contg. a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson-Crick polar edge of thymine or 5hmU via the O2, N3 and O4 atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivs. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C1' of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.
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    Maiti, A., Noon, M. S., MacKerell, A. D., Jr., Pozharski, E., and Drohat, A. C. (2012) Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA Proc. Natl. Acad. Sci. U.S.A. 109, 8091– 8096
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    Cadet, J. and Wagner, J. R. (2014) TET enzymatic oxidation of 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine Mutat. Res., Genet. Toxicol. Environ. Mutagen. 764–765, 18– 35
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    TET enzymatic oxidation of 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine
    Cadet, Jean; Wagner, J. Richard
    Mutation Research, Genetic Toxicology and Environmental Mutagenesis (2014),764-765 (),18-35CODEN:MRGMFI; ISSN:1383-5718. (Elsevier B.V.)
    A review. 5-Methylcytosine and methylated histones have been considered for a long time as stable epigenetic marks of chromatin involved in gene regulation. This concept has been recently revisited with the detection of large amts. of 5-hydroxymethylcytosine, now considered as the sixth DNA base, in mouse embryonic stem cells, Purkinje neurons and brain tissues. The dioxygenases that belong to the ten eleven translocation (TET) oxygenase family have been shown to initiate the formation of this Me oxidn. product of 5-methylcytosine that is also generated although far less efficiently by radical reactions involving hydroxyl radical and one-electron oxidants. It was found as addnl. striking data that iterative TET-mediated oxidn. of 5-hydroxymethylcytosine gives rise to 5-formylcytosine and 5-carboxylcytosine. This survey focuses on chem. and biochem. aspects of the enzymic oxidn. reactions of 5-methylcytosine that are likely to be involved in active demethylation pathways through the implication of enzymic deamination of 5-methylcytosine oxidn. products and/or several base excision repair enzymes. The high biol. relevance of the latter modified bases explains why major efforts have been devoted to the design of a broad range of assays aimed at measuring globally or at the single base resoln., 5-hydroxymethylcytosine and the two other oxidn. products in the DNA of cells and tissues. Another crit. issue that is addressed in this review article deals with the assessment of the possible role of 5-methylcytosine oxidn. products, when present in elevated amts. in cellular DNA, in terms of mutagenesis and interference with key cellular enzymes including DNA and RNA polymerases.
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    Maiti, A., Michelson, A. Z., Armwood, C. J., Lee, J. K., and Drohat, A. C. (2013) Divergent mechanisms for enzymatic excision of 5-formylcytosine and 5-carboxylcytosine from DNA J. Am. Chem. Soc. 135, 15813– 15822
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    Divergent Mechanisms for Enzymatic Excision of 5-Formylcytosine and 5-Carboxylcytosine from DNA
    Maiti, Atanu; Michelson, Anna Zhachkina; Armwood, Cherece J.; Lee, Jeehiun K.; Drohat, Alexander C.
    Journal of the American Chemical Society (2013),135 (42),15813-15822CODEN:JACSAT; ISSN:0002-7863. (American Chemical Society)
    5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidn. of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair (BER). Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2'-deoxycytidine ionizes with pKa values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a monoanion at physiol. pH. Calcns. do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that N-glycosidic bond hydrolysis follows a stepwise (SN1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, i.e., the leaving group quality of the target base. Calcns. here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid-catalyzed, and the pH profile indicates that caC ionizes in the enzyme-substrate complex with an apparent pKa of 5.8, likely at N3. Mutational anal. reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells.
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    Wossidlo, M., Arand, J., Sebastiano, V., Lepikhov, K., Boiani, M., Reinhardt, R., Scholer, H., and Walter, J. (2010) Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes EMBO J. 29, 1877– 1888
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    Zhang, P., Su, L., Wang, Z., Zhang, S., Guan, J., Chen, Y., Yin, Y., Gao, F., Tang, B., and Li, Z. (2012) The involvement of 5-hydroxymethylcytosine in active DNA demethylation in mice Biol. Reprod. 86, 104
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    The involvement of 5-hydroxymethylcytosine in active DNA demethylation in mice
    Zhang, Peng; Su, Li; Wang, Zhongwei; Zhang, Sheng; Guan, Jiyu; Chen, Yue; Yin, Yupeng; Gao, Fei; Tang, Bo; Li, Ziyi
    Biology of Reproduction (2012),86 (4),104/1-104/9CODEN:BIREBV; ISSN:0006-3363. (Society for the Study of Reproduction)
    Active DNA demethylation occurs after a sperm enters an egg. However, the mechanisms for the active DNA demethylation remain poorly understood. Ten-eleven translocation enzymes were recently shown to catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Thus, we decided to investigate the role of 5hmC in active demethylation. We analyzed the methylation and hydroxymethylation status in metaphase II oocytes as well as 1-cell stage and cleavage stage embryos. In zygotes, 5hmC was mainly detected in the paternal pronucleus and it increased from the pronuclear-2 (PN2) to PN5 stages, an indication that 5hmC was involved in paternal genomic DNA demethylation. Bisulfite-sequencing PCR and qGluMS-PCR (DNA glucosylation and digestion before quant. PCR) results showed that a large redn. of methylcytosine and hydroxymethylcytosine in LINE1 (long interspersed nuclear element 1) occurred between the 4- and 8-cell stages, which indicates that demethylation potentially occurred after the 4-cell stage. We then microinjected mouse zygote with plasmids that were methylated in vitro by SssI methylase and analyzed for the hydroxymethylation status of the plasmids promoter region. We found that the rapid onset of expression of the unmethylated plasmids in mouse embryos happened in <12 h, but the expression of methylated plasmids was delayed until 50 h when most embryos were at the 8-cell stage. Quant. GluMS-PCR results suggested that 5hmC was present in the plasmid's promoter region at the MspI site where the active demethylation occurred. Our results demonstrate that 5hmC is involved in active demethylation in mice.
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    Kriaucionis, S. and Heintz, N. (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain Science 324, 929– 930
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    The nuclear DNA base 5-Hydroxymethylcytosine is present in Purkinje neurons and the brain
    Kriaucionis, Skirmantas; Heintz, Nathaniel
    Science (Washington, DC, United States) (2009),324 (5929),929-930CODEN:SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Despite the importance of epigenetic regulation in neurol. disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, the authors detected the presence of an unusual DNA nucleotide. Here, using TLC, HPLC, and mass spectrometry, the authors identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). The hmdC was a constituent of nuclear DNA that was highly abundant in the brain, suggesting a role in epigenetic control of neuronal function. The hmdC was found to constitute 0.6% of the total nucleotides in Purkinje cells, 0.2% in granule cells, and was not present in cancer cell lines.
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    Munzel, M., Lischke, U., Stathis, D., Pfaffeneder, T., Gnerlich, F. A., Deiml, C. A., Koch, S. C., Karaghiosoff, K., and Carell, T. (2011) Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine Chemistry 17, 13782– 13788
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    Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine
    Munzel Martin; Lischke Ulrike; Stathis Dimitrios; Pfaffeneder Toni; Gnerlich Felix A; Deiml Christian A; Koch Sandra C; Karaghiosoff Konstantin; Carell Thomas
    Chemistry (Weinheim an der Bergstrasse, Germany) (2011),17 (49),13782-8 ISSN:.
    5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.
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    Simmons, J. M., Muller, T. A., and Hausinger, R. P. (2008) Fe(II)/alpha-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism Dalton Trans. 5132– 5142
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    Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., Agarwal, S., Iyer, L. M., Liu, D. R., Aravind, L., and Rao, A. (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1 Science 324, 930– 935
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    Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1
    Tahiliani, Mamta; Koh, Kian Peng; Shen, Yinghua; Pastor, William A.; Bandukwala, Hozefa; Brudno, Yevgeny; Agarwal, Suneet; Iyer, Lakshminarayan M.; Liu, David R.; Aravind, L.; Rao, Anjana
    Science (Washington, DC, United States) (2009),324 (5929),930-935CODEN:SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-Me group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
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    Ito, S., D’Alessio, A. C., Taranova, O. V., Hong, K., Sowers, L. C., and Zhang, Y. (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification Nature 466, 1129– 1133
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    Bienvenu, C., Wagner, J. R., and Cadet, J. (1996) Photosensitized oxidation of 5-methyl-2′-deoxycytidine by 2-methyl-1,4-naphthoquinone: characterization of 5-(hydroperoxymethyl)-2′-deoxycytidine and stable methyl group oxidation products J. Am. Chem. Soc. 118, 11406– 11411
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    Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. B., Swenberg, J. A., He, C., and Zhang, Y. (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine Science 333, 1300– 1303
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    Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine
    Ito, Shinsuke; Shen, Li; Dai, Qing; Wu, Susan C.; Collins, Leonard B.; Swenberg, James A.; He, Chuan; Zhang, Yi
    Science (Washington, DC, United States) (2011),333 (6047),1300-1303CODEN:SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    5-Methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) proteins. Here, we show that, in addn. to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymic activity-dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse embryonic stem cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivs. in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidn. followed by decarboxylation.
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    Pfaffeneder, T., Hackner, B., Truss, M., Munzel, M., Muller, M., Deiml, C. A., Hagemeier, C., and Carell, T. (2011) The discovery of 5-formylcytosine in embryonic stem cell DNA Angew. Chem., Int. Ed. 50, 7008– 7012
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    The Discovery of 5-Formylcytosine in Embryonic Stem Cell DNA
    Pfaffeneder, Toni; Hackner, Benjamin; Truss, Matthias; Muenzel, Martin; Mueller, Markus; Deiml, Christian A.; Hagemeier, Christian; Carell, Thomas
    Angewandte Chemie, International Edition (2011),50 (31),7008-7012, S7008/1-S7008/18CODEN:ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We provide here direct evidence for the presence of 5-formylcytosine (fC) in DNA isolated from mouse embryonic stem cells (mES) and mouse embryoid body (mEB) cells. The fC levels were found to dramatically decrease with ongoing differentiation. Interestingly, we do not detect the fC compd. in DNA isolated from neuronal cells, which contain the highest amts. of 5-hydroxymethyl cytosine (hmC). We explain this result on the basis of data from a recent study by Song and co-workers who showed that active demethylation in adult brain cells proceeds likely through deamination of hmC to 5-hydroxymethyl uridine (hmU) followed by removal of the hmU base by the base excision repair pathway. Thus, fC is in this respect a clear marker nucleoside for the development of mES cells. It has not escaped our notice that the oxidative demethylation of methylcytosine via 5-formylcytosine we have postulated, immediately suggests a possible globally acting epigenetic control mechanism.
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    Raiber, E., Murat, P., Chirgadze, D. Y., Beraldi, D., Luisi, B. F., and Balasubramanian, S. (2015) 5-Formylcytosine alters the structure of the DNA double helix Nat. Struct. Mol. Biol. 22, 44– 49 DOI: 10.1038/nsmb.2936
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    5-Formylcytosine alters the structure of the DNA double helix
    Raiber, Eun-Ang; Murat, Pierre; Chirgadze, Dimitri Y.; Beraldi, Dario; Luisi, Ben F.; Balasubramanian, Shankar
    Nature Structural & Molecular Biology (2015),22 (1),44-49CODEN:NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase. Genomics and proteomics studies have suggested an addnl. role for 5fC in transcription regulation through chromatin remodeling. Here we propose that 5fC might affect these processes through its effect on DNA conformation. Biophys. and structural anal. revealed that 5fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those comprising other cytosine modifications. The 1.4-Å-resoln. X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shows how 5fC changes the geometry of the grooves and base pairs assocd. with the modified base, leading to helical underwinding.
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    Site-specific DNA damage recognition by enzyme-induced base flipping
    Stivers, James T.
    Progress in Nucleic Acid Research and Molecular Biology (2004),77 (),37-65CODEN:PNMBAF; ISSN:0079-6603. (Elsevier)
    A review discussing enzyme-induced base flipping. It particularly discusses why base flipping has evolved as the sole mechanism for recognition and catalysis by DNA glycosylases, and the natures of the energetic barriers that an enzyme must overcome to extricate a base from its position in the DNA duplex. It also describes the temporal events that occur during enzymic base flipping and the steps that contribute to catalytic specificity. Some of the new approaches to investigate base flipping are also described.
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    Hardeland, U., Bentele, M., Lettieri, T., Steinacher, R., Jiricny, J., and Schar, P. (2001) Thymine DNA glycosylase Prog. Nucleic Acid Res. Mol. Biol. 68, 235– 253
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    Thymine DNA glycosylase
    Hardeland, Ulrike; Bentele, Marc; Lettieri, Teresa; Steinacher, Roland; Jiricny, Josef; Schar, Primo
    Progress in Nucleic Acid Research and Molecular Biology (2001),68 (),235-253CODEN:PNMBAF; ISSN:0079-6603. (Academic Press)
    A review. More than 50% of colon cancer-assocd. mutations in the p53 tumor suppressor gene are C→T transitions. The majority of them locate in CpG dinucleotides and are thought to have arisen through spontaneous hydrolytic deamination of 5-methylcytosine. This deamination process gives rise to G·T mispairs that need to be repaired to G·C in order to avoid C→T mutation. Similarly, deamination of cytosine generates G·U mispairs that also produce C→T transitions if not repaired. Restoration of both G·T and G·U mismatches was shown to be mediated by a short-patch excision repair pathway, and one principal player implicated in this process may be thymine DNA glycosylase (TDG). Human TDG was discovered as an enzyme that has the potential to specifically remove thymine and uracil bases mispaired with guanine through hydrolysis of their N-glycosidic bond, thereby generating abasic sites in DNA and initiating a base excision repair reaction. The same protein was later found to interact phys. and functionally with the retinoid receptors RAR and RXR, and this implicated an unexpected function of TDG in nuclear receptor-mediated transcriptional activation of gene expression. The objective of this chapter is to put together the results of different lines of experimentation that have explored the thymine DNA glycosylase since its discovery and to critically evaluate their implications for possible physiol. roles of this enzyme. (c) 2001 Academic Press.
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    Hashimoto, H., Zhang, X., and Cheng, X. (2013) Selective excision of 5-carboxylcytosine by a thymine DNA glycosylase mutant J. Mol. Biol. 425, 971– 976
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    Selective Excision of 5-Carboxylcytosine by a Thymine DNA Glycosylase Mutant
    Hashimoto, Hideharu; Zhang, Xing; Cheng, Xiaodong
    Journal of Molecular Biology (2013),425 (6),971-976CODEN:JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    The mammalian thymine DNA glycosylase (TDG) excises the mismatched base, uracil, thymine or 5-hydroxymethyluracil (5hmU), as well as removes 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) when paired with a guanine. In the previously solved structure of TDG in complex with DNA contg. 5caC, the side chain of asparagine 157 (N157) contacts the 5-carboxyl moiety of 5caC via a weak hydrogen bond. We examd. the role of N157 in recognition of 5caC by mutagenesis. The asparagine-to-alanine (N157A) mutant has no detectable base excision activity for a G:T mismatch, and its excision activity is reduced for other substrates including G:5caC. Unexpectedly, the asparagine-to-aspartate (N157D) mutant has a comparable base excision rate for G:5caC substrate to that of wild type, but it only has residual activity for G:U and no detectable activity for other substrates. We further show that the N157D mutant has higher activity for 5caC at a lower pH (6.0), suggesting that increased protonation of the carboxylate of 5caC and the aspartate facilitates base excision. The N157D mutant remains highly specific for 5caC even in the presence of large excess of genomic DNA, a property that can potentially be used for mapping the very low amt. of 5caC in genomes.
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    Proceedings of the National Academy of Sciences of the United States of America (1981),78 (4),2179-83CODEN:PNASA6; ISSN:0027-8424.
    The crystal structure of the synthetic DNA dodecamer d(CpGpCpGpApApTpTpCpGpCpG) was refined to a residual error of R = 17.8% at 1.9-Å resoln. (2-σ data). The mol. forms slightly >1 complete turn of right-handed double-stranded B helix. The 2 ends of the helix overlap and interlock minor grooves with neighboring mols. up and down a 21 screw axis, producing a 19° bend in helix axis over the 11-base-pair steps of the dodecamer. In the center of the mol., where perturbation is least, the helix has a mean rotation of 36.9° per step, or 9.8 base pairs per turn. The mean propeller twist (total dihedral angle between base planes) between A·T base pairs in the center of the mol. is 17.3°, and that between C·G pairs on the 2 ends avs. 11.5°. Individual deoxyribose ring conformations, measured by the C5'-C4'-C3'-O3' torsion angle δ, exhibit an approx. Gaussian distribution centered around the C1'-exo position with δav. = 123° and a range of 79-157°. Purine sugars cluster at high δ values, and pyrimidine sugars cluster at lower δ. A tendency toward 2-fold symmetry in sugar conformation about the center of the mol. is detectable in spite of the destruction of ideal 2-fold symmetry by the mol. bending. More strikingly, sugar conformations of paired bases appear to follow a principle of anticorrelation, with δ values lying approx. the same distance to either side of the center value, δ = 123°. This same anticorrelation is also obsd. in other DNA and DNA·RNA structures.
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    The NMR Structure of a DNA Dodecamer in an Aqueous Dilute Liquid Crystalline Phase
    Tjandra, Nico; Tate, Shin-ichi; Ono, Akira; Kainosho, Masatsune; Bax, Ad
    Journal of the American Chemical Society (2000),122 (26),6190-6200CODEN:JACSAT; ISSN:0002-7863. (American Chemical Society)
    The soln. structure of the DNA dodecamer d(CGCGAATTCGCG)2 has been studied in an aq. liq. cryst. medium contg. 5% w/v bicelles. These phospholipid particles impose a small degree of orientation on the DNA duplex mols. with respect to the magnetic field and permit the measurement of dipolar interactions. Expts. were carried out on several samples with different isotopic labeling patterns, including two complementary samples, in which half of the nucleotides were uniformly enriched with 13C and deuterated at the H2''and H5' positions. From this, 198 13C-1H and 10 15N-1H one-bond dipolar coupling restraints were derived, in addn. to 200 approx. 1H-1H dipolar coupling and 162 structurally meaningful NOE restraints. Although loose empirical restraints for the phosphodiester backbone torsion angles were essential for obtaining structures that satisfy all exptl. data, they do not contribute to the energetic penalty function of the final minimized structures. Except for addnl. regular Watson-Crick hydrogen bond restraints and std. van der Waals and electrostatic terms used in the mol. dynamics-based structure calcn., the structure is detd. primarily by the dipolar couplings. The final structure is highly regular, without any significant bending or kinks, and with C2'-endo/C1'-exo sugar puckers corresponding to regular B-form DNA. Most local parameters, including sugar puckers, glycosyl torsion angles, and propeller twists, are also tightly detd. by the NMR data. The precision of the detd. structures is limited primarily by the uncertainty in the exact magnitude and rhombicity of the alignment tensor. This causes considerable spread in parameters such as the degree of base-pair opening and the width of the minor groove, which are relatively sensitive to the alignment tensor values used.
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    Singh, S. K., Szulik, M. W., Ganguly, M., Khutsishvili, I., Stone, M. P., Marky, L. A., and Gold, B. (2011) Characterization of DNA with an 8-oxoguanine modification Nucleic Acids Res. 39, 6789– 6801
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    Characterization of DNA with an 8-oxoguanine modification
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    Nucleic Acids Research (2011),39 (15),6789-6801CODEN:NARHAD; ISSN:0305-1048. (Oxford University Press)
    The oxidn. of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidn. product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodn. stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodn. studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol-1 over a 16-116 mM NaCl range) due to a significant redn. in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration, indicating that the environment around an oxoG·C is fundamentally different than that found at G·C. The temp.-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG·C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.
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    UV absorption provides the nearly universal basis for detg. concns. of nucleic acids. Values for the UV extinction coeffs. of DNA and RNA rely on the mononucleotide values detd. 30-50 yr ago. We show that nearly all of the previously published extinction coeffs. for the nucleoside-5'-monophosphates are too large, and in error by as much as 7%. Concns. based on complete hydrolysis and the older set of values are too low by ∼4% for typical RNA and 2-3% for typical DNA samples. We also analyzed data in the literature for the extinction coeffs. of unpaired DNA oligomers. Robust prediction of concns. can be made using 38 μg/A260 unit for single-stranded DNA (ssDNA) having non-repetitive sequences and 40-80% GC. This is superior to currently used predictions that account for nearest-neighbor frequency or base compn. The latter result in concns. that are 10-30% too low for typical ssDNA used as primers for PCR and other similar techniques. Methods are described here to accurately measure concns. of nucleotides by NMR. NMR can be used to accurately det. concns. (and extinction coeffs.) of biomols. within 1%.
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    The general forms of the equations required to ext. thermodn. data from equil. transition curves on oligomeric and polymeric nucleic acids of any molecularity, were derived. Significantly, since the equations and protocols are general, they also can be used to characterize thermodynamically equil. processes in systems other than nucleic acids. How the reduced forms of the general equations have been used by many investigators to evaluate mono- and bimol. transitions was discussed. How these equations can be generalized to calc. thermodn. parameters from common exptl. observables for transitions of higher molecularities was also explained. The strengths and weaknesses of each method of data anal. were emphasized so that investigators can select the approach most appropriate for their exptl. circumstances. How to analyze calorimetric heat capacity curves and noncalorimetric differentiated melting curves is described so as to ext. both model-independent and model-dependent thermodn. data for transitions of any molecularity. The general equations and methods of anal. described should be of particular interest to labs. that currently are investigating assocn. and dissocn. processes in nucleic acids that exhibit molecularities greater than two.
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    Two solvent-suppressing NMR pulse sequences are described which do not generate a linear phase shift and are therefore free from the corresponding baseline errors. The first sequence (90°y;τ;90°-y or jump and return) cancels the solvent line to first order and provides strictly const. phase, apart from a 180° step change at the solvent frequency. The second (set, jump and return) is derived from "jump and return" to provide second-order cancellation, at the price of modest phase variations. Both sequences use only strong nonselective pulses, at the solvent frequency. Strong-pulse sequences are easier to design and implement, and they are insensitive to drift in the pulse amplitude.
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    A review. The 19ID undulator beamline of the Structure Biol. Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 Å wavelength) with fluxes up to 8-18 × 1012 photons s-1 (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-max.) at the sample position can be varied from 2.2 mm × 1.0 mm (horizontal × vertical, unfocused) to 0.083 mm × 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a κ-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallog. expts., thus maximizing the chances of their success. A large-area mosaic 3 × 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallog. expts. are presented.
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    HKL-3000: the integration of data reduction and structure solution - from diffraction images to an initial model in minutes
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    A new approach that integrates data collection, data redn., phasing and model building significantly accelerates the process of structure detn. and on av. minimizes the no. of data sets and synchrotron time required for structure soln. Initial testing of the HKL-3000 system (the beta version was named HKL-2000_ph) with more than 140 novel structure detns. has proven its high value for MAD/SAD expts. The heuristics for choosing the best computational strategy at different data resoln. limits of phasing signal and crystal diffraction are being optimized. The typical end result is an interpretable electron-d. map with a partially built structure and, in some cases, an almost complete refined model. The current development is oriented towards very fast structure soln. in order to provide feedback during the diffraction expt. Work is also proceeding towards improving the quality of phasing calcn. and model building.
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    Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-redn. package XDS are explained, including the new features of automatic detn. of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the detn. of correction/scaling factors.
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    The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
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    The various phys. factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analyzed to set the real resoln. of the data set, to detect bad regions (e.g. bad images), to analyze radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation anal. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be detd. from scores such as correlation coeffs. between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.
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    Macromol. crystallog. is an iterative process. Rarely do the first crystals provide all the necessary data to solve the biol. problem being studied. Each step benefits from experience learned in previous steps. To monitor the progress, the HKL package provides 2 tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), are provided, and the Bayesian reasoning and multicomponent error model facilitates obtaining the proper error ests. and (2) visualization of the process plays a double role by helping the operator to confirm that the process of data redn., including the resulting statistics, is correct, and allowing one to evaluate problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and redn. has been reached. At that point, the effort should be directed to solving the structure. The methods presented here have been applied to solve a large variety of problems, from inorg. mols. with 5 Å unit cell to rotavirus of 700 Å diam. crystd. in 700 × 1000 × 1400 Å cell. Overall quality of the method was tested by many researchers by successful application of the programs to MAD structure detns.
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    MOLREP is an automated program for mol. replacement which uses effective new approaches in data processing and rotational and translational searching. These include an automatic choice of all parameters, scaling by Patterson origin peaks and soft resoln. cutoff. One of the cornerstones of the program is an original full-symmetry translation function combined with a packing function. Information from the model already placed in the cell is incorporated in both translation and packing functions. A no. of tests using exptl. data proved the ability of the program to find the correct soln. in difficult cases.
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    Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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    One of the most important aspects of macromol. structure refinement is the use of prior chem. knowledge. Bond lengths, bond angles and other chem. properties are used in restrained refinement as subsidiary conditions. This contribution describes the organization and some aspects of the use of the flexible and human/machine-readable dictionary of prior chem. knowledge used by the max.-likelihood macromol.-refinement program REFMAC5. The dictionary stores information about monomers which represent the constitutive building blocks of biol. macromols. (amino acids, nucleic acids and saccharides) and about numerous org./inorg. compds. commonly found in macromol. crystallog. It also describes the modifications the building blocks undergo as a result of chem. reactions and the links required for polymer formation. More than 2000 monomer entries, 100 modification entries and 200 link entries are currently available. Algorithms and tools for updating and adding new entries to the dictionary have also been developed and are presented here. In many cases, the REFMAC5 dictionary allows entirely automatic generation of restraints within REFMAC5 refinement runs.
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