Proofreading exonuclease activity of human DNA polymerase delta and its effects on lesion-bypass DNA synthesis.
Replicative DNA polymerases possess 3'' --> 5'' exonuclease activity to reduce misincorporation of incorrect nucleotides by proofreading during replication. To examine if this proofreading activity modulates DNA synthesis of damaged templates, we constructed a series of recombinant human DNA polymerase delta (Pol delta) in which one or two of the three conserved Asp residues in the exonuclease domain are mutated, and compared their properties with that of the wild-type enzyme. While all the mutant enzymes lost more than 95% exonuclease activity and severely decreased the proofreading activity than the wild-type, the bypass efficiency of damaged templates was varied: two mutant enzymes, D515V and D402A/D515A, gave higher bypass efficiencies on templates containing an abasic site, but another mutant, D316N/D515A, showed a lower bypass efficiency than the wild-type. All the enzymes including the wild-type inserted an adenine opposite the abasic site, whereas these enzymes inserted cytosine and adenine opposite an 8-oxoguanine with a ratio of 6:4. These results indicate that the exonuclease activity of human Pol delta modulates its intrinsic bypass efficiency on the damaged template, but does not affect the choice of nucleotide to be inserted.
PubMed ID: 19282447
PMC ID: PMC2685094
Article link: Nucleic Acids Res.
Grant support: CA06315 NCI NIH HHS , CA06927 NCI NIH HHS , CA092584 NCI NIH HHS
Genes referenced: pcna slc19a1
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|Figure 1. Expression and purification of recombinant human Pol δ. (A) Schematic structures of expression plasmids for Pol δ. (B) Purification profile of Pol δ. Each fraction was electrophoresed in an SDS 4–12% polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, load to a Ni2+-Sepharose column; lane 2, flow-through of the Ni2+-Sepharose column; lane 3, a wash fraction with 1 M NaCl-containing buffer; lane 4, a wash fraction with 100 mM imidazole-containing buffer; lane 5, eluate from the Ni2+-Sepharose column with 300 mM imidazole-containing buffer; lane 6, flow-through of an SP-Sepharose column, lane 7, a wash fraction with 200 mM NaCl-containing buffer; lane 8, a wash fraction with 500 mM NaCl-containing buffer; lane 9, eluate from the SP column with 1 M NaCl-containing buffer. (C) Purified Pol δ wild-type (wt) and exonuclease mutant enzymes. (D) Purified recombinant human RFC visualized with Coomassie staining.|
|Figure 2. Sequences of oligonucleotide primers and templates. 3′-Terminal mispaired nucleotides in primers are underlined. Lesions in templates, 8-OG (O) and tetrahydrofuran AP site analog (F), are also underlined. 5′-Terminal biotin is indicated as B. The lac operator sequence (lacO) is indicated by shading. Numbering of the sequence is based on the primer strand.|
|Figure 3. Primer extension on an end-blocked oligonucelotide by recombinant PCNA, RFC and Pol δ. (A) Schematic structure of the end-blocked primer/template. A biotin (B) is attached at the 5′ end of the template and is bound to a streptavidin (SA)-conjugated magnetic bead. (B) Primer extension reactions were conducted with indicated protein factors and the normal primer/49-nt G template as described in ‘Materials and Methods’ section.|
|Figure 4. Complementation of Xenopus egg extracts with recombinant Pol δ. Single-stranded circular DNA was annealed with non-radioactive ssDNA primer (Figure 2) and used for replication synthesis in the presence of 32P-dATP. (A) DNA replication in Pol δ- or mock-depleted NPE. Samples taken at the indicated times were treated with SDS/EDTA/Proteinase K and separated by 1% TAE/agarose gel electrophoresis. (B) Rescue of the replication defect by the recombinant Pol δ.|
|Figure 5. Site-directed mutants of Pol δ exonuclease domains. (A) Amino-acid sequence comparison of the exonucelase domains of replicative DNA polymerases. Budding yeast and human Pol δ catalytic subunits are compared to DNA polymerases from bacterophages, RB69 and T4. Identical residues and similar residues conserved among the four enzymes are indicated in red and blue fonts, respectively. Three aspartic acids presumed to be critical for exonuclease activity are indicated with asterisks. Mutations introduced into human Pol δ in this study are indicated below. (B) DNA polymerase activity with wild-type and three mutant human Pol δ enzymes. The assay was conducted with the normal primer/49-nt G templates in the presence of PCNA, RFC and the Lac repressor as described in ‘Materials and Methods’ section. The activity was calculated as [intensity of the fully extended product]/([intensity of the fully extended product] + [intensity of the unextended primer]), and normalized with the activity of the wild-type enzyme as 100%. Average activities and standard deviations of mutant enzymes from three experiments were presented in the right panel. (C) Exonuclease assay of Pol δ. 5′-Labeled single-stranded oligonucleotide (ssDNA primer in Figure 2) was incubated with indicated Pol δ enzymes. The exonuclease activity was calculated as described in ‘Materials and Methods’ section. Average activities and standard deviations from three reactions were presented in the right panel.|
|Figure 6. Extension of 3′-mispaired primers. Reactions were conducted with indicated primers annealed to the 49-nt G template (Figure 2) in the absence or presence of dNTPs as described in ‘Materials and Methods’ section. All the reactions contained PCNA, RFC and Lac repressor.|
|Figure 7. Nucleotide determination of products extended from 3′-mispaired primers. (A) Representative data of pyrosequencing. The ratio of the corrected nucleotide and the mispaired nucleotide was obtained from two peaks in the pyrogram (boxed). In this case with the 49 nt A:G primer, A is the mispaired nucleotide, while C is the corrected nucleotide opposite G. (B) Summary of pyroseqencing data. Efficiency of synthesis of fully extended products over unextended primers (quantitated from Figure 6) is presented along with the ratio of proofread products (dark area) and mispaired products (gray area). Deletion products from the 57-nt G:G primer were indicated as white area. (C) Three mechanisms for extension of the 57-nt G:G primer. The 3′-mispaired nucleotide is indicated with an asterisk.|
|Figure 8. Primer extension by Pol δ on lesion-containing templates. (A) Extension on the 49-nt X templates. Positions of the primer end and the lesion (X) are indicated on the right of the gel. (B) Extension on the 63-nt X templates. Both reactions were conducted in the presence of PCNA, RFC and Lac repressor as described in ‘Materials and Methods’ section.|
|Figure 9. Bypass efficiency of Pol δ on damaged templates. The efficiency was calculated as [intensity of the full extended product]/([intensity of the fully extended product] + [intensity of the lesion-stalled product]) from the data shown in Figure 8. The efficiency on the normal templates was calculated with the intensity of the products stopped at the position corresponding to the lesions.|
|Figure 10. Nucleotides incorporated opposite the lesions. Pyrosequencing of the fully extended products on the 63-nt X templates (Figure 8B) was conducted as described in ‘Materials and Methods’ section. The pyrograms shown here are corresponding to the position opposite the lesions of damaged templates or the control G nucleotide of the normal template.|