Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence - PubMed (original) (raw)

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Gintautas Tamulaitis et al. Nucleic Acids Res. 2007.

Abstract

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI-DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.

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Figures

Figure 1.

Figure 1.

Flipped nucleotides in the Ecl18kI–DNA complex structure (2FQZ). (A) General view of the Ecl18kI dimer–DNA complex. Protein is shown in spacefill. Residues 60–69 and 91–136 are removed for clarity. DNA is depicted in red. (B) Binding ‘pocket’ for the flipped out base. A flipped adenine base is accommodated in the ‘pocket’ made by the side chain atoms of Arg57 on one face and the indole ring of Trp61 on the other face.

Figure 2.

Figure 2.

Gel mobility shift analysis of the interactions between Ecl18kI and oligoduplexes. (A) Ecl18kI binding of the oligoduplex III containing the recognition sequence. (B) Ecl18kI binding of the cognate oligoduplex I containing 2-AP instead of the central A base. The binding reactions contained 33P-labeled 25 bp oligoduplex (0.1 nM) and the Ecl18kI at concentrations as indicated by each lane. Samples were analyzed by PAGE under non-denaturing conditions (see, Material and Methods Section). Gels were run in the presence of 5 mM of Ca2+ ions.

Figure 3.

Figure 3.

Fluorescence study of base flipping by Ecl18kI in solution. Titration of 250 nM 2-AP containing oligoduplex I with increasing amounts of Ecl18kI in the presence (A) and absence (D) of Ca2+ ions, respectively. Corrected 2-AP emission spectra of Ecl18kI–DNA complexes (1250 nM Ecl18kI and 250 nM oligoduplexes I or II) in the presence (B) and absence (E) of Ca2+ ions (see Materials and Methods Section for the details). Diagrams in (C) and (F) illustrate the maximum fluorescence intensity values of the corrected fluorescence emission spectra presented in (B) and (E), respectively.

Figure 4.

Figure 4.

2-AP fluorescence in the ternary complexes of the wt Ecl18kI and W61A mutant. Diagrams illustrate the fluorescence intensity values of the corrected fluorescence emission spectra at fluorescence maximum (367 nm for wt, 365 nm for W61A and 370 nm for oligoduplexes). Reactions contained 1250 nM protein and 250 nM oligoduplexes I or II and were measured in the presence of Ca2+ ions.

Figure 5.

Figure 5.

Ecl18kI, EcoRII and PspGI monomer structures. Central β-sheets are colored in red. N-terminal effector domain which is unique to EcoRII (44), is shown in gray. PspGI structural model (26) is shown.

Figure 6.

Figure 6.

2-AP fluorescence in the ternary complexes of EcoRII-C, PspGI and MvaI. Diagrams show the fluorescence intensity values of the corrected fluorescence emission spectra at fluorescence maximum (360 nm for EcoRII-C and PspGI, 370 nm for MvaI and oligoduplexes). Reactions contained 1250 nM protein and 250 nM oligoduplexes I or II and were measured in the presence of Ca2+ ions.

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References

    1. Klimasauskas S, Kumar S, Roberts RJ, Cheng X. HhaI methyltransferase flips its target base out of the DNA helix. Cell. 1994;76:357–369. - PubMed
    1. Reinisch KM, Chen L, Verdine GL, Lipscomb WN. The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell. 1995;82:143–153. - PubMed
    1. Goedecke K, Pignot M, Goody RS, Scheidig AJ, Weinhold E. Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog. Nat. Struct. Biol. 2001;8:121–125. - PubMed
    1. Horton JR, Liebert K, Hattman S, Jeltsch A, Cheng X. Transition from nonspecific to specific DNA interactions along the substrate-recognition pathway of dam methyltransferase. Cell. 2005;121:349–361. - PMC - PubMed
    1. Horton JR, Liebert K, Bekes M, Jeltsch A, Cheng X. Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase. J. Mol. Biol. 2006;358:559–570. - PMC - PubMed

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