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Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.

Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

International audience; The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.

Generation of 3′ single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5′ strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3′-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.

Summary The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways—MMS2-dependent template switch and Pol ζ-dependent bypass—are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1–4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome., Graphical Abstract Highlights ► Unprocessed rNMPs in the chromosomes sensitize cells to replication stress ► Postreplication repair pathways are essential to tolerating rNMP-containing templates ► DNA polymerase ζ efficiently bypasses rNMPs in DNA templates ► Postreplication repair is chronically activated in RNase H1/H2 defective cells

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends., Author Summary Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.

The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.