Palm mutants in DNA polymerases alpha and eta alter DNA replication fidelity and translesion activity.
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 26000-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.
Niimi
Atsuko
A
Division of Molecular Carcinogenesis, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
Limsirichaikul
Siripan
S
Yoshida
Shonen
S
Iwai
Shigenori
S
Masutani
Chikahide
C
Hanaoka
Fumio
F
Kool
Eric T
ET
Nishiyama
Yukihiro
Y
Suzuki
Motoshi
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004249:DNA Damage; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D029701:Saccharomyces cerevisiae Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases