Human DNA polymerase h (hPolh) is one of the newly identi®ed Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPolh accurately bypasses UV-associated cis±syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the ®ngers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identi®es Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPolh S62G, a mutant polymerase with enhanced activity, to bypass ®ve other site-speci®c lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.
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