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Papers In Press, published online ahead of print March 7, 2003
Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya 466-8550
Corresponding Author: msuzuki{at}med.nagoya-u.ac.jp
Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol alpha mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn948 or Tyr951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis, but ~6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys944 or Gly952 were not viable, which is consistent with the observation that mutants with substitutions at Gly952 have strongly reduced catalytic activity in vitro. Gly952 may provide a space for the nascent base pair, and thus may play an essential function in Saccharomyces cerevisiae DNA pol alpha. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.
J. Biol. Chem, 10.1074/jbc.M208605200
Submitted on August 22, 2002
Revised on February 5, 2003
Accepted on March 7, 2003
Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha
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