Translesion replication by DNA polymerase delta depends on processivity accessory proteins and differs in specificity from DNA polymerase beta.
Mutations caused by DNA damage lead to the development of cancer. The critical step in the formation of these mutations is the replication of unrepaired lesions in DNA by DNA polymerases, a process termed translesion replication. Using a newly developed method for preparation of gapped plasmids, containing a site-specific synthetic abasic site, we analyzed translesion replication with purified mammalian DNA polymerases delta and beta. DNA polymerase delta was found to be unable to replicate through the abasic site. Addition of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication. Thus, similar to Escherichia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions. Under comparable conditions, DNA polymerase beta was unable to bypass the abasic site, unless its concentration was greatly increased. Analysis of translesion replication products revealed a marked difference in the specificity of bypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme involved insertion of a dAMP residue opposite the abasic site, DNA polymerase beta tended to skip over the abasic site, producing mainly minus frameshifts (73%). The significance of these results for in vivo translesion replication is discussed.
Daube
S S
SS
Department of Biological Chemistry, Faculty of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Tomer
G
G
Livneh
Z
Z
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D019951:DNA Polymerase beta