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Molecular and Cellular Biology, April 2004, p. 2734-2746, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2734-2746.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Palm Mutants in DNA Polymerases {alpha} and {eta} Alter DNA Replication Fidelity and Translesion Activity

Atsuko Niimi,1 Siripan Limsirichaikul,1 Shonen Yoshida,1,{dagger} Shigenori Iwai,2 Chikahide Masutani,3 Fumio Hanaoka,3,4 Eric T. Kool,5 Yukihiro Nishiyama,6 and Motoshi Suzuki1*

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 {alpha} that were associated with a defect in error discrimination. Among them, L868F DNA polymerase {alpha} has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase {alpha}. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase {alpha}-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase {alpha} 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 {eta}, and the F34L mutant of S. cerevisiae DNA polymerase {eta} has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase {alpha} 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.


* Corresponding author. Mailing address: Division of Molecular Carcinogenesis, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. Phone: 81 52 744 2456. Fax: 81 52 744 2457. E-mail: msuzuki{at}med.nagoya-u.ac.jp.

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


Molecular and Cellular Biology, April 2004, p. 2734-2746, Vol. 24, No. 7
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.7.2734-2746.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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