Characterization of the Two Small Subunits of Saccharomyces cerevisiae DNA Polymerase δ

Abstract

Yeast DNA polymerase ␦ (Pol␦) has three subunits of 125, 58, and 55 kDa. The gene for the 125-kDa catalytic subunit (POL3) has been known for several years. Here we describe the cloning of the genes for the 58-and 55-kDa subunits using peptide sequence analysis and searching of the yeast genome data base. The 58-kDa subunit, encoded by the POL31 gene, shows 23-28% sequence similarity to the 48-kDa subunit of human Pol␦ and to S. pombe Cdc1. POL31 is allelic to HYS2 and SDP5. The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base). Very limited sequence similarity was observed between Pol32p and Schizosaccharomyces pombe Cdc27, the functionally analogous subunit in S. pombe Pol␦. The POL32 gene is not essential, but a deletion mutant shows cold sensitivity for growth and is sensitive to hydroxyurea and DNA damaging agents. In addition, lethality was observed when the POL32 deletion mutation was combined with conditional mutations in either the POL3 or POL31 gene. Pol32⌬ strains are weak antimutators and are defective for damage-induced mutagenesis. The POL32 gene product binds proliferating cell nuclear antigen. A gel filtration analysis showed that Pol32p is a dimer in solution. When POL31 and POL32 were co-expressed in Escherichia coli, a tetrameric (Pol31p⅐Pol32p) 2 species was detected by gel filtration, indicating that the two subunits form a complex. DNA polymerase ␦ (Pol␦)1 is the major replicative DNA polymerase in the eukaryotic cell. This insight is based on extensive in vitro studies using the simian 40 virus as a model system, and on genetic and biochemical studies in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe (reviewed in Refs. 1 and 2). These genetic studies have not only shown a role for Pol␦ in bulk DNA replication but also for maintaining genome fidelity via the proofreading exonuclease activity of this enzyme (3, 4). A similar, but perhaps less defined role has also been identified for DNA polymerase ⑀, whereas the synthetic function of DNA polymerase ␣-primase appears to be limited to that of initiator RNA-DNA synthesis for priming Okazaki fragments on the lagging strand of the DNA replication fork (5-7).The best characterized Pol␦ from mammalian cells is the enzyme purified from fetal calf thymus tissue, a heterodimer with a catalytic subunit of 125 kDa and a second subunit of 48 kDa (8). The small subunit is required for efficient stimulation of the polymerase processivity by the proliferating cell nuclear antigen (PCNA) (9, 10).The forms of Pol␦ isolated from bakers' and fission yeast are more complex. Our previous studies of S. cerevisiae Pol␦ indicated that the enzyme might consist of three or more subunits (11). Very recently, Pol␦ has been isolated from S. pombe as an enzyme with five distinct subunits, four of which also appear to be subunits based on genetic arguments (12).In this paper we describe an improved purification of S. cerevisiae Pol␦ as a three-subunit enzyme, the cloning of the two small subunits of Pol␦, and a genetic and ...

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

“…Yeast two-hybrid assays, particularly with yeast genes, can be misleading because false positives can result from a bridging interaction through a third yeast protein, as for instance was observed with the third subunit of Pol ␦. Pol32 appeared to interact with itself in a two-hybrid analysis, but this interaction was actually mediated by proliferating cell nuclear antigen (18,29). A second line of evidence for a dimeric Pol ⑀ was based upon gel filtration experiments (19).…”

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…S. cerevisiae Pol d is a heterotrimer consisting of the p125 and p50 homologs (Pol3 and Pol31, respectively) and, a third subunit, Pol32 [Gerik et al, 1998]. S. cerevisiae Pol d will be referred to as yPold3 to distinguish it from its counterpart in Schizosaccharomyces pombe, which has a fourth subunit, Cdm1 [Bermudez et al, 2002], and which will be referred to as yPold4.…”

Crystallization and preliminary crystallographic analysis of the complex of the second and third regulatory subunits of human Pol δ

“…Using a two-hybrid screening, human p66 has been shown to contain p50-binding and PCNA-binding domains within the 144 N-terminal and 20 C-terminal amino acids, respectively (Pohler et al, 2005). Studies with the model organism Saccharomyces cerevisiae (baker's yeast) revealed that many of the essential functions of Pol , including its involvement in break-induced replication (BIR; Lydeard et al, 2007) and the mutagenic bypass pathway (Gerik et al, 1998;Huang et al, 2000Huang et al, , 2002Gibbs et al, 2005), depend on interactions between pol31 and pol32, which are the yeast orthologues of human p50 and p66, respectively. Interestingly, only the pol31-binding domain of pol32 (Johansson et al, 2004) was essential and sufficient for the contribution of Pol to BIR (Lydeard et al, 2007).…”