Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha

J Biol Chem. 2003 May 23;278(21):19071-8. doi: 10.1074/jbc.M208605200. Epub 2003 Mar 7.


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 Asn-948 or Tyr-951 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 approximately 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 Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. 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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence*
  • Base Pairing
  • Binding Sites
  • Catalysis
  • Conserved Sequence*
  • DNA Polymerase I / chemistry*
  • DNA Polymerase I / genetics
  • DNA, Fungal / biosynthesis
  • DNA, Fungal / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Gene Deletion
  • Gene Library
  • Glycine / genetics
  • Kinetics
  • Lysine / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Mutagenesis, Site-Directed
  • Mutation
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Alignment
  • Structure-Activity Relationship
  • Taq Polymerase / chemistry


  • DNA, Fungal
  • Deoxyguanine Nucleotides
  • 2'-deoxyguanosine 5'-phosphate
  • Taq Polymerase
  • DNA Polymerase I
  • Lysine
  • Glycine