Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System (original) (raw)
Related papers
Reconstitution of Human DNA Polymerase δ Using Recombinant Baculoviruses
Journal of Biological Chemistry, 2001
Eukaryotic DNA polymerase ␦ is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase ␦. However, rigorous purification of human or bovine DNA polymerase ␦ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase ␦, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The foursubunit DNA polymerase ␦ displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase ␦ efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase ␦.
Nucleic Acids Research, 1998
protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine-5′-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer∨template either by human DNA polymerase α primase (polα), human DNA polymerase β (polβ) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primertemplate in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3′-end of the primer, while the p14 subunit locates relatively far from the 3′-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.
Journal of Biological Chemistry, 2009
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol ␦, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol  and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling. Downloaded from FIGURE 3. Fen-1 inhibits the synthesis of products beyond the abasic site by DNA polymerase ␦ but is essential for efficient strand displacement-dependent bypass of the abasic site by DNA polymerase . The reactions were performed as described under "Experimental Procedures." A, 60 nM DNA pol ␦ were incubated for the indicated times on the Gap-3 AP template in the presence of 110 nM PCNA alone (lanes 2-5) or in combination with 13 nM (as trimer) RP-A (lanes 6 -9) or 2.7 nM recombinant human Fen-1 (lanes 10 -13) or both . B, 60 nM DNA pol ␦ (lanes 1-8) or 30 nM DNA pol  (lanes 9 -24), were incubated with 110 nM PCNA alone (lanes 1-8 and 17-24) or in combination with 2.7 nM Fen-1 (lanes 5-8 and 21-24), in the absence (lanes 4, 8, 20, and 24) or in the presence , and 21-23) of increasing amounts of RP-A. Lanes 9 -16, RP-A titration in the presence of 30 nM DNA pol  in the absence of PCNA and in the absence (lanes 9 -12) or in the presence (lanes 13-16) of 2.7 nM Fen-1.
Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase
DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8-10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn 2 + but not Mg 2 + significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn 2 + provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.
The p12 Subunit of Human Polymerase δ Modulates the Rate and Fidelity of DNA Synthesis
Biochemistry, 2010
This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Pol δ) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the foursubunit complex, the three-subunit complex lacking p12, and site-directed mutants of each lacking proofreading exonuclease activity. Results show that p12 modulates the rate and fidelity of DNA synthesis by Pol δ. All four complexes synthesize DNA in a rapid burst phase and a slower, more linear phase. In the presence of p12, the burst rates of DNA synthesis are ∼5 times faster, while the affinity of the enzyme for its DNA and dNTP substrates appears unchanged. The p12 subunit alters Pol δ fidelity by modulating the proofreading 3 0 to 5 0 exonuclease activity. In the absence of p12, Pol δ is more likely to proofread DNA synthesis because it cleaves single-stranded DNA twice as fast and transfers mismatched DNA from the polymerase to the exonuclease sites 9 times faster. Pol δ also extends mismatched primers 3 times more slowly in the absence of p12. Taken together, the changes that p12 exerts on Pol δ are ones that can modulate its fidelity of DNA synthesis. The loss of p12, which occurs in cells upon exposure to DNA-damaging agents, converts Pol δ to a form that has an increased capacity for proofreading.
ø29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates
Journal of Molecular Biology, 1998
A 44 kDa C-terminal fragment of ù29 DNA polymerase has been separately expressed and puri®ed from Escherichia coli cells. As expected, the truncated protein lacked the 3 H-5 H exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of ù29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn 2 as metal activator, although the catalytic ef®ciency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by ù29 DNA polymerase (>70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of ù29 DNA replication. We conclude that the C-terminal portion of ù29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more speci®c functions (TP-priming, high processivity and strand-displacement) inherent to ù29 DNA polymerase.
Journal of Biological Chemistry, 2004
Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol. These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.
Nucleic Acids Research, 2004
The human nuclear single-stranded (ss) DNAbinding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein±DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5¢protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a`zerolength' photoreactive group (4-thio-2¢-deoxyuridine-5¢-monophosphate) embedded in the 3¢ end of the DNA primer, or a group connected to the 3¢ end by a lengthy linker (5-{N-[N-(4-azido-2,5-di¯uoro-3chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2¢-deoxyuridine-5¢-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C´p32D´p14) was capable of correctly recognizing the primer± template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D´p14) was incapable of detectably recognizing the DNAjunction structures, suggesting an orchestrating role for p70C in this process.
Primer initiation and extension by T7 DNA primase
The EMBO Journal, 2006
T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a twofold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.