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 forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Genotoxic treatments in human cells consistently induce uncoupling of replication forks and their remodeling into four-way junctions by the RAD51 recombinase.

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 and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

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 leading and lagging strand replication at irreparable UV lesions, thus generating long ssDNA regions on one side of the fork. Furthermore, small ssDNA gaps accumulate along replicated duplexes, likely resulting from repriming events downstream of the lesions on both leading and lagging strands. Translesion synthesis and homologous recombination counteract gap accumulation, without affecting fork progression. The DNA damage checkpoint contributes to gap repair and maintains a replication-competent fork structure. We propose that the coordinated action of checkpoint, recombination, and translesion synthesis-mediated processes at the fork and behind the fork preserves the integrity of replicating chromosomes by allowing efficient replication restart and filling the resulting ssDNA gaps.

The role of Rad51 in an unperturbed cell cycle has been difficult to dissect from its DNA repair function. Here, using electron microscopy (EM) to visualize replication intermediates (RIs) assembled in Xenopus laevis egg extract we show that Rad51 is required to prevent the accumulation of ssDNA gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading and lagging strand DNA synthesis. Instead, ssDNA gaps behind forks, which are exacerbated on damaged templates, result from Mre11 dependent degradation of newly synthesized DNA strands as they can be suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct and unanticipated roles for Rad51 at replication forks demonstrating that Rad51 protects newly synthesised DNA from Mre11 dependent degradation and promotes continuous DNA synthesis.