Budding yeast Rtt107 prevents checkpoint hyperactivation after replicative stress by limiting DNA damage

Cells respond to DNA damage by activating cell cycle checkpoints, arresting cell division or DNA replication while damage is repaired. In Saccharomyces cerevisiae, activation of the checkpoint kinase Rad53 leads to cell cycle arrest, with Rad53 deactivation required for proper resumption of the cell cycle. Rtt107 is a S. cerevisiae protein that acts as a scaffold in the response to DNA damage, and rtt107Δ mutants exhibit prolonged activation of Rad53 when subjected to replication stress. This phenotype has been attributed to checkpoint dampening, wherein an Rtt107-Slx4-Dpb11 interaction limits formation of a Rad9-Dpb11 complex that promotes Rad53 activation. However, we found that the rtt107Δ mutant contains higher levels of DNA damage during replication stress, presenting an alternative possible cause of Rad53 hyperactivation. We therefore sought to address the relevance of checkpoint dampening to the Rad53 hyperactivation phenotype of the rtt107Δ mutant by using a rad9-ST462,474AA allele that specifically disrupts Rad9-Dpb11 interaction. Incorporation of the rad9-ST462,474AA allele slightly suppressed the rtt107Δ mutant's DNA damage sensitivity phenotypes, while having little effect on Rad53 hyperactivation. This indicated that in the context of acute replication stress, Rad53 hyperactivation in the rtt107Δ mutant did not primarily result from Rad9-Dpb11 interaction. A H2A-S129A mutation, which generally reduces Rad9-mediated Rad53 activation, led to more robust suppression of rtt107Δ mutant phenotypes. Suppression of rtt107Δ mutant DNA damage sensitivity by the H2A-S129A or the rad9-ST462,474AA alleles required intact DNA damage tolerance pathways, indicating a reliance of the rtt107Δ mutant on tolerance pathways for reasons other than misregulation of Rad53 activity. Collectively, this work proposed a revised model of Rad53 hyperactivation after acute replicative stress in the rtt107Δ mutant, in which this phenotype was primarily a consequence of excess DNA damage.