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In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged(More)
Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the(More)
DNA replication forks pause in front of lesions on the template, eventually leading to cytotoxic chromosomal rearrangements. The in vivo structure of damaged eukaryotic replication intermediates has been so far elusive. Combining electron microscopy (EM) and two-dimensional (2D) gel electrophoresis, we found that UV-irradiated S. cerevisiae cells uncouple(More)
The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in(More)
S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by(More)
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(More)
The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks(More)
O(6)-Methylguanine ((Me)G) is a highly cytotoxic DNA modification generated by S(N)1-type methylating agents. Despite numerous studies implicating DNA replication, mismatch repair (MMR), and homologous recombination (HR) in (Me)G toxicity, its mode of action has remained elusive. We studied the molecular transactions in the DNA of yeast and mammalian cells(More)
The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new(More)
Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use(More)