Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperaturesensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 39 / 59 proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 39 / 59 exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.
SummaryMost of the prokaryotic and eukaryotic replicative polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells.
KeywordsDNA polymerase; Pol epsilon holoenzyme (Pol ε HE); Spontaneous mutagenesis; Fidelity of DNA replication; Protein-protein interaction; DPB2 (YPR175W) and POL2 (YNL262W) genes of the yeast Saccharomyces cerevisiae
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