The four-subunit DNA polymerase a-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We Cloning of the S. cerevisiae POLl, PRII, and PR12 genes, which encode the p180, p48, and p58 subunits of the yeast complex, respectively, and the study ofpoll, pril, and pn2 lethal and conditional alleles have been essential in establishing the roles of the corresponding gene products in mitotic DNA replication and in identifying functional domains in the polymerase and primase polypeptides (8,26,29,42,51,54). Moreover, poll, pnil, and pn2 conditional mutants are defective in premeiotic DNA synthesis and show an enhanced rate of intrachromosomal recombination and spontaneous mutation, which is generally correlated with the severity of their defects in cell growth and DNA synthesis (9,41,42). Since these mutant strains fail to accumulate high-molecular-weight DNA products (9, 29), the formation of nicked and gapped replicated DNA molecules might be responsible for the hyperrecombination phenotype usually found in yeast DNA synthesis mutants (33).No enzymatic activity has been found to be associated with the 86-kDa protein species (B subunit), which is tightly bound to the p180 polypeptide, and the physiological role of p86 is presently unknown (7). In vitro reconstitution studies with purified components indicate that p86 is not required for polymerase-primase interaction, and its presence does not change the catalytic properties of Pol a (6, 7). Recently, it has been proposed that the human p86 homolog might serve as a molecular tether during DNA replication, because this subunit mediates the in vitro interaction between the human Pol a-primase complex and T antigen bound to the SV40 origin of replication (14).As a first step in identifying the physiological role of the B subunit of the yeast Pol a-primase complex in vivo, we have mutagenized by the two-codon insertion mutagenesis method (3) the essential POL12 gene, which was recently found to encode the p86 polypeptide (7, 36a). The analysis of 923

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpointdependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

A 5600-base-pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A poll temperature-sensitive mutation, encoding a DNA polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity. The DNA polymerase-primase complex from Saccharomyces cerevisiae has been purified to near homogeneity (2), and specific antibodies have been used to isolate the genes encoding DNA polymerase I (3-5) and the 48-kDa subunit of DNA primase (p48) (6). Both genes are unique in the haploid yeast genome and perform essential functions (4-6). The availability of the cloned DNA polymerase I gene has allowed the production, by in vitro mutagenesis, of temperature-sensitive mutants that will be extremely useful to establish the role ofthis enzyme in DNA metabolism (7,8).Moreover, direct analysis of POLl mRNA showed that the amount of DNA polymerase I mRNA fluctuates during the cell cycle and meiosis and that DNA polymerase I mRNA is induced after DNA damage (9). The finding that the expression of the POLI gene is under cell-cycle control leads to questions about the nature of the cis-and trans-acting genetic elements that regulate the expression of this essential gene.To meaningfully address such questions, the nucleotide sequence of the DNA polymerase I gene and of its putative upstream regulatory region is required. Moreover, the nucleotide sequence will permit calculation of the molecular weight of the POLI encoded protein and identification of possible functional domains as homologies with other DNA polymerase sequences are found and as poll mutants are characterized. We have determined the nucleotide sequence of the POLl genet and have identified regions of homology between the yeast DNA polymerase polypeptide and DNA polymerases from animal viruses and bacteriophages. We also report the sequence of one point mutation, poll-l (8), that confers a temperature-sensitive phenotype by altering the stability of the DNA polymerase-primase complex and that possibly defines a functional domain involved in protein-protein interactions. MATERIALS AND METHODSPlasmids and Strains. Plasmids, pAP70, pAP33, and pAP1, were constructed by cloning, respectively, the 3.4-kilobase (kb) Sal I segment, in both orientations, and the 2.2-kb Sal I-BamHI segment (Fig. 1) into pGEM4 (Promega Biotec, Madison, WI). The two restriction fragments, derived from plasmid pGL7-4 (4), span the entire POLl coding sequence plus 700 base pairs (bp) of the 5'-noncoding region that are sufficient for cell viability (8). Plasmid DNA was digested with Kpn I and BamHI (p...

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFAI gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase a-primase complex and with DNA polymerase 8.The identification of eukaryotic replication proteins has relied mainly on the use of in vitro replication systems. For example, the simian virus 40 system allowed the isolation of a number of replication proteins from human cell extracts and the reconstitution of an in vitro replication system with purified components (10,31,52,55). However, the answer to the question of whether these proteins function in vivo to replicate chromosomal DNA requires the production of mutations in the corresponding genes and the characterization of the obtained mutant strains. The use of the yeast Saccharomyces cerevisiae has been particularly useful for this purpose (8,9).The human single-stranded-DNA-binding protein (also known as replication protein A or replication factor A [RF-A]), originally identified as a protein factor required for simian virus 40 DNA replication in vitro, is a heterotrimer of 70-, 34-, and 11-kDa subunits (19,57,58). The p70 polypeptide is sufficient for DNA binding (6,32,59), while the function of the p34 and p1l subunits is still unknown. The p34 polypeptide is phosphorylated in a cell cycle-dependent manner (14,17,20,22), suggesting that RF-A might be a target of the regulatory mechanisms driving the G1-to-S phase transition during the cell cycle.The RF-A complex is highly conserved in all eukaryotes (6,39,58), and the three RF-A genes in budding yeast cells are essential for cell viability (7). Nevertheless, yeast RF-A only partially substitutes for human RF-A in the in vitro replication of simian virus 40 (6), indicating that species-specific interactions between RF-A and other replication proteins are important for its biological activity. Similarly, the DNA polymerase ct (pol ao)-primase complex is involved in determining the species specificity of the DNA replication process (42,47,48). The RF-A and pol o-primase complexes are quite peculiar, since these two protein complexes are required during both the initiation and elongation steps of DNA replication (11,53,55,56). In vitro studies indicate that they interact with each other as well as with T antigen (15,16,37,41,43), suggesting a pivotal role for these proteins in DNA replication. Moreover, the function of the RF-A complex might not be limited to the replication process, since human RF-A seems to be required for the nucleotide excision repair of UV-damaged DNA in vitro (12), and both yeas...