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.
Fazlieva
Ruzaliya
R
Medical Science Division, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Spittle
Cynthia S
CS
Morrissey
Darlene
D
Hayashi
Harutoshi
H
Yan
Hong
H
Matsumoto
Yoshihiro
Y
D000818:Animals; D020137:Base Pair Mismatch; D004247:DNA / Q000096:biosynthesis*; D004249:DNA Damage; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010063:Ovum / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D014982:Xenopus laevis
D009711:Nucleotides; D011994:Recombinant Proteins; D004247:DNA; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases