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

Abstract:

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.

Polymerases:

Topics:

Source / Purification, Fidelity, Nucleotide Incorporation, Historical Protein Properties (MW, pI, ...)

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Sce Pol alpha Y951P Ogawa M2003 Cloned or native Cloned in yeast
Sce Pol alpha Y951P Ogawa M2003 Tagged No
Sce Pol alpha Y951P Ogawa M2003 Nucleotide Substitution Rate 0.0034 errors/bp Technique: Forward mutational
Sce Pol alpha Y951P Ogawa M2003 Full length or truncated Full length
Sce Pol alpha Y951P Ogawa M2003 Processivity 2bp
Sce Pol alpha Ogawa M2003 Cloned or native Cloned in yeast
Sce Pol alpha Ogawa M2003 Tagged No
Sce Pol alpha Ogawa M2003 Nucleotide Substitution Rate 0.0034 errors/bp Technique: Forward mutational
Sce Pol alpha Ogawa M2003 Full length or truncated Full length
Sce Pol alpha Ogawa M2003 Processivity 12bp
Sce Pol alpha Ogawa M2003 Specific Activity 2.8E+04 units/mg Technique: Polymerase Assay (calf thymus DNA)

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