The DNA replication checkpoint response stabilizes stalled replication forks

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Abstract
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 repair1,2,3,4,5. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase3,4,6. Active Rad53 slows DNA synthesis when DNA is damaged7 and prevents firing of late origins of replication8,9. Further, rad53 mutants are unable to recover from a replication block10. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes6,11,12,13. 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 technique14 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.