Obstacles such as DNA damage can block the progression of DNA replication forks. This is a major source of genome instability that can lead to cell transformation or death. The budding yeast MMS1 and MMS22 genes were identified in a screen for mutants that were hypersensitive to DNA alkylation that blocks replisome progression. I set out to investigate the cellular roles of these genes and found that cells lacking MMS1 or MMS22 are hypersensitive to a wide variety of genotoxins that stall or block replication forks, and are severely defective in their ability to recover from DNA alkylation damage. Homologous recombination (HR) is an important mechanism for the rescue of stalled or blocked replication forks and for the repair of double-strand breaks (DSBs). Strikingly, MMS1 and MMS22 are required for HR induced by replication stress but not by DSBs, and the underlying mechanisms were explored.I next identified the uncharacterized protein C6ORF167 (MMS22L) as a putative human Mms22 orthologue. MMS22L interacts with NF?BIL2/TONSL, the histone chaperone ASF1 and subunits of the MCM replicative helicase. MMS22L colocalizes with TONSL at perturbed replication forks and at sites of DNA damage. MMS22L and TONSL are important for the repair of collapsed replication forks as depletion of MMS22L or TONSL from human cells causes DNA damage during S–phase and hypersensitivity to agents that cause fork collapse. These defects are consistent with the observations that MMS22L and TONSL are required for the efficient loading of the RAD51 recombinase onto resected DNA ends and for efficient HR. These data indicate that MMS22L and TONSL are novel regulators of genome stability that enable efficient HR.

Mms1 and Mms22 are important mediators of genome stability in Saccharomyces cerevisiae. Deletion mutants of both genes display decreased viability in the presence of a number of known DNA damaging agents as well as agents that perturb DNA replication. Here I present a detailed analysis of Mms1 and Mms22 function in stabilizing DNA replication forks. I find that mms1∆ and mms22∆ strains accumulate spontaneous DNA damage and have an increased rate of mutation during a normal cell cycle. Additionally, treatment with genotoxic agents causes defects in induced recombination as well as inhibiting the ability of cells to efficiently recover from DNA damage. Genetic interaction data support a model where Mms1 and Mms22 function with components of the replication fork. Accordingly I observed that the enrichment of key replication fork proteins at regions proximal to replication origins is decreased in mms1∆ and mms22∆ cells during replication stress. Furthermore, I monitored DNA replication in wild-type and mms1∆ strains and found that mms1∆ strains exhibit irregular fork progression under conditions of replication stress. I therefore concluded that Mms1 and Mms22 function at replication forks. Lastly, I uncovered that Mms1 is regulated by Rtt101-dependent ubiquitin-mediated proteolysis. Mms1 degradation is likely necessary for its function as overexpression of Mms1 results in decreased cell viability both during a normal cell cycle, and during treatment with DNA damaging agents. I found that Mms1 and Mms22 operate at sites of DNA replication to promote the stability of the genome, especially under conditions of DNA damage and replication fork stress.

Mms1 and Mms22 are important mediators of genome stability in Saccharomyces cerevisiae. Deletion mutants of both genes display decreased viability in the presence of a number of known DNA damaging agents as well as agents that perturb DNA replication. Here I present a detailed analysis of Mms1 and Mms22 function in stabilizing DNA replication forks. I find that mms1∆ and mms22∆ strains accumulate spontaneous DNA damage and have an increased rate of mutation during a normal cell cycle. Additionally, treatment with genotoxic agents causes defects in induced recombination as well as inhibiting the ability of cells to efficiently recover from DNA damage. Genetic interaction data support a model where Mms1 and Mms22 function with components of the replication fork. Accordingly I observed that the enrichment of key replication fork proteins at regions proximal to replication origins is decreased in mms1∆ and mms22∆ cells during replication stress. Furthermore, I monitored DNA replication in wild-type and mms1∆ strains and found that mms1∆ strains exhibit irregular fork progression under conditions of replication stress. I therefore concluded that Mms1 and Mms22 function at replication forks. Lastly, I uncovered that Mms1 is regulated by Rtt101-dependent ubiquitin-mediated proteolysis. Mms1 degradation is likely necessary for its function as overexpression of Mms1 results in decreased cell viability both during a normal cell cycle, and during treatment with DNA damaging agents. I found that Mms1 and Mms22 operate at sites of DNA replication to promote the stability of the genome, especially under conditions of DNA damage and replication fork stress.