The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G 1 requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase α-primase complex in response to intra-S DNA damage.

No abstract

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

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.