Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha.
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.
Ogawa
Masanori
M
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
Limsirichaikul
Siripan
S
Niimi
Atsuko
A
Iwai
Shigenori
S
Yoshida
Shonen
S
Suzuki
Motoshi
M
D000595:Amino Acid Sequence; D020029:Base Pairing; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D017353:Gene Deletion; D015723:Gene Library; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D019914:Taq Polymerase / Q000737:chemistry
D004271:DNA, Fungal; D003848:Deoxyguanine Nucleotides; C007257:2'-deoxyguanosine 5'-phosphate; D019914:Taq Polymerase; D004256:DNA Polymerase I; D008239:Lysine; D005998:Glycine