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 criti cal step in the formation of these mutations is the replication of unrepair ed lesions in DNA by DNA polymerases, a process termed translesion replicat ion. Using a newly developed method for preparation of gapped plasmids, con taining a site-specific synthetic abasic site, we analyzed translesion repl ication with purified mammalian DNA polymerases delta and beta. DNA polymer ase delta was found to be unable to replicate through the abasic site. Addi tion of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication. Thus, similar to Esche richia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions. Under comparable condition s, to bypass blocking DNA polymerase beta was unable to bypass the abasic s ite, unless its concentration was greatly increased. Analysis of translesio n replication products revealed a marked difference in the specificity of b ypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme invo lved insertion of a dAMP residue opposite the abasic site, DNA polymerase b eta tended to skip over the abasic site, producing mainly minus frameshifts (73%). The significance of these results for in vivo translesion replicati on is discussed.