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Palm Mutants in DNA Polymerases and Alter DNA Replication Fidelity and Translesion Activity

Atsuko Niimi,¹ Siripan Limsirichaikul,¹ Shonen Yoshida,^1, Shigenori Iwai,² Chikahide Masutani,³ Fumio Hanaoka,^3,4 Eric T. Kool,⁵ Yukihiro Nishiyama,⁶ and Motoshi Suzuki^1*

Division of Molecular Carcinogenesis, Center for Neural Disease and Cancer,¹ Department of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550,⁶ Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531,² Graduate School of Frontier Biosciences, Osaka University, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Suita, Osaka 565-0871,³ Cellular Physiology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan,⁴ Department of Chemistry, Stanford University, Stanford, California 94305⁵

Received 8 September 2003/ Returned for modification 18 October 2003/ Accepted 11 December 2003

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase that were associated with a defect in error discrimination. Among them, L868F DNA polymerase has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase . In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase -dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3' T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase , and the F34L mutant of S. cerevisiae DNA polymerase has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Present address: Kaikokai Rehabilitation Hospital, Amagun, Aichi 490-1405, Japan.

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