用复制抑制剂羟基脲处理酵母细胞激活S期检查点激酶Rad53,引发阻断DNA复制起点发射的反应,稳定复制叉,并防止有丝分裂纺锤体的过早延伸。我们以前发现Stn1是端粒结合Cdc13-Stn1-Ten1复合物的一个亚基,绕过羟基脲中的Rad53检查点功能,即使Rad53被正常激活,也会引起晚起源点火和过早的主轴伸展。这里,我们显示,与RAD53的损失相比,Stn1的过度生产通过非常相似的途径起作用,汇聚在MCM复合物上,该复合物引发了起始发射并形成了复制性DNA解旋酶的催化核心。首先,影响Mcm2和Mcm5的突变阻断了Stn1过度生产破坏S期检查点的能力。第二,功能缺失stn1突变补偿rad53S阶段检查点缺陷。第三Stn1过量产生抑制Mcm7中的突变。第四,stn1突变体在非端粒基因组位置积累单链DNA,强制要求复制后DNA修复。我们根据一个模型来讨论这些相互作用,在该模型中,Stn1充当辅助复制因子,可促进ORI处的MCM激活,并可能在通过具有挑战性的模板推进的复制叉处保持MCM活动。
Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.