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 ...

Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation. Replication factor C (RFC)1 is the eukaryotic clamp loader that uses the energy of ATP hydrolysis to load the replication clamp proliferating cell nuclear antigen (PCNA) onto the DNA at a primer-template junction. RFC in yeast is a heteropentamer that consists of a large subunit (Rfc1, 95 kDa) and four small subunits (Rfc2-Rfc5, 36 -40 kDa). The yeast Rfc subunits are all quite similar to each other at the amino acid level (24 -37%) and to the human Rfc subunits. However, each yeast subunit shares the highest degree of sequence homology with the analogous subunit from human RFC (reviewed in Ref. 1). The sequence similarity is localized to the N-terminal half of the subunits in regions termed RFC boxes II-VIII. Most notable are the putative ATP-binding motifs in boxes III and V. The amino acids in box III, the Walker A motif, create a characteristic fold, the P loop, which forms a pocket for binding of the ␤-and ␥-phosphates of ATP (2, 3).Our biochemical studies reported in the second paper (4) of this series show that four ATP molecules can bind to RFC when PCNA and primer-template DNA are also present. This coincides with our current understanding of the primary sequence determinants of ATP-binding domains, which indicates that four of the five subunits comprising the RFC complex contain consensus ATP-binding motifs. (see Fig. 1A). The Rfc5 subunit lacks critical residues in both the A and B motifs and therefore this domain may have a more structural function analogous to the ␦Ј subunit of Escherichia coli DNA polymerase III holoenzyme (5).Mutation of the conserved lysine residue to glutamic acid in...