Proofreading exonuclease activity of human DNA polymerase δ and its effects on lesion-bypass DNA synthesis |
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Authors: | Ruzaliya Fazlieva Cynthia S. Spittle Darlene Morrissey Harutoshi Hayashi Hong Yan Yoshihiro Matsumoto |
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Affiliation: | 1.Medical Science Division, 2.Cancer Biomarker and Genotyping Facility and 3.Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA |
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Abstract: | 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 δ (Pol δ) 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 δ modulates its intrinsic bypass efficiency on the damaged template, but does not affect the choice of nucleotide to be inserted. |
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