Academic journal article Genetics

Dpb2p, a Noncatalytic Subunit of DNA Polymerase [Epsilon], Contributes to the Fidelity of DNA Replication in Saccharomyces Cerevisiae

Academic journal article Genetics

Dpb2p, a Noncatalytic Subunit of DNA Polymerase [Epsilon], Contributes to the Fidelity of DNA Replication in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

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 temperature-sensitive 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 3' [arrow right] 5' 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 3' [arrow right] 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.

UNDERSTANDING of the mechanisms that control the generation of mutations on normal and damaged DNA templates is crucial in studies of carcinogen-esis and mutagenesis. The mutator hypothesis for the origins of cancer suggests that both early and late stages of tumor progression are connected to expression of a mutator phenotype (Loeb 2001; Loeb et al. 2003; Bielas et al. 2006). One likely source of spontaneous mutations are errors occurring during DNA replication. Thus, understanding of the mechanisms controlling DNA replication fidelity has become a major challenge of current molecular biology.

The accuracy of DNA synthesis ismaintained by three highly conserved processes: correct base selection by the DNA polymerases, removal of base insertion errors by 3' [arrow right] 5' exonucleolytic proofreading activity of DNA polymerases, and postreplication correction of poly-merase errors by the DNA mismatch repair system (MMR). Thus, DNA polymerases are central to replication fidelity. Most of the major replicative DNA poly-merases, often called replicases, are multi-subunit holoenzymes (HE). In eukaryotic cells, DNA replication is executed by at least three DNA polymerases: Pol a, Pol d, and Pol e. Pol e HE is a four-subunit complex composed of aDNA polymerase/exonuclease subunit, Pol2p, and three auxiliary subunits: Dpb2p, Dpb3p, and Dpb4p (Hamatake et al. 1990; Dua et al. 2000; Chilkova et al. 2003; Asturias et al. 2006). This composition of subunits is conserved from yeast to humans (Kesti et al. 1993; Li et al. 1997; Jokela et al. 1998; Li et al. 2000; Feng et al. 2003; Spiga and D'Urso 2004). Two subunits, Pol2p and Dpb2p, are essential in yeast (Morrison et al. 1990; Araki et al. 1991a). Dpb3p and Dpb4p are not essential (Araki et al. 1991b; Ohya et al. 2000). Pol2p is essential for leading-strand DNA replication and plays a role in linking DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae (Araki et al. 1992; Budd and Campbell 1993; Navas et al. 1995, 1996; Dua et al. 1998, 1999; Pursell et al. 2007). Strains carrying a temperature-sensitivemutation in the DPB2 gene (dpb2-1) suggest that Dpb2p is also essential for DNA replication (Araki et al. 1991a). At the restrictive temperature, dpb2-1 cells are dumbbell shaped, which is characteristic for a DNA replication defect. Dpb2p interacts with Pol2p (Sugino 1995; Dua et al. 2000) and is phosphorylated by Cdc28p, a cyclin-dependent protein kinase, in a cellcycle dependent manner (Kesti et al. 2004). Nevertheless, the precise cellular function of Dpb2p is unknown.

Strains carrying exonuclease-deficient Pol d (pol3-01) or Pol e (pol2-4) are mutators. Using URA3 forward mutation or his7-2 reversion assays, Morrison and Sugino (1994) demonstrated that the pol3-01 allele ele vates spontaneous-mutation rates by ~100-fold, as compared to an only 10-fold increase for the pol2-4 allele. …

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