In this study, researchers investigated how the Mec1/Rad53 checkpoint pathway regulates DNA replication fork progression through damaged DNA in budding yeast. The Mec1 and Rad53 checkpoint kinases are essential for maintaining cell viability in the presence of DNA-damaging agents or stalled replication forks. They are thought to function by inhibiting cell cycle progression, allowing time for DNA repair. However, the study found that the DNA-alkylation agent methyl methanesulphonate (MMS) significantly reduces the rate of DNA replication fork progression, but this moderation does not require Rad53 or Mec1. Instead, the accelerated S phase in checkpoint mutants is primarily due to inappropriate initiation events. Wild-type cells can complete DNA replication in the presence of MMS, while replication forks in checkpoint mutants collapse irreversibly. The cytotoxicity of MMS in checkpoint mutants occurs specifically when cells enter S phase with DNA damage, suggesting that preventing damage-induced DNA replication fork catastrophe is a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation. The study also found that MMS slows S phase progression in checkpoint-proficient yeast strains, which may be due to the inhibition of late origin firing or a reduced rate of replication fork progression. The results suggest that the Mec1/Rad53 checkpoint pathway plays a critical role in regulating DNA replication fork progression through damaged DNA, ensuring the survival of cells under DNA damage conditions.In this study, researchers investigated how the Mec1/Rad53 checkpoint pathway regulates DNA replication fork progression through damaged DNA in budding yeast. The Mec1 and Rad53 checkpoint kinases are essential for maintaining cell viability in the presence of DNA-damaging agents or stalled replication forks. They are thought to function by inhibiting cell cycle progression, allowing time for DNA repair. However, the study found that the DNA-alkylation agent methyl methanesulphonate (MMS) significantly reduces the rate of DNA replication fork progression, but this moderation does not require Rad53 or Mec1. Instead, the accelerated S phase in checkpoint mutants is primarily due to inappropriate initiation events. Wild-type cells can complete DNA replication in the presence of MMS, while replication forks in checkpoint mutants collapse irreversibly. The cytotoxicity of MMS in checkpoint mutants occurs specifically when cells enter S phase with DNA damage, suggesting that preventing damage-induced DNA replication fork catastrophe is a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation. The study also found that MMS slows S phase progression in checkpoint-proficient yeast strains, which may be due to the inhibition of late origin firing or a reduced rate of replication fork progression. The results suggest that the Mec1/Rad53 checkpoint pathway plays a critical role in regulating DNA replication fork progression through damaged DNA, ensuring the survival of cells under DNA damage conditions.