复制叉失速时,叉后形成的RPA包被的单链DNA(ssDNA)激活共济失调毛细血管扩张突变和Rad3相关(ATR)激酶,同时启动依赖Rad18的PCNA单项化。然而,这两个事件之间是否存在串扰以及这种相互作用的潜在生理意义仍然难以捉摸。在这项研究中,我们证明了在复制应激期间,ATR在Ser403上磷酸化人Rad18,Ser403是Rad18中先前未鉴定的PIP基序(PCNA相互作用肽)的相邻残基。这个磷酸化事件破坏了Rad18和PCNA之间的相互作用,从而限制了Rad18介导的PCNA单尿素化的程度。因此,肿瘤抑制蛋白SLX4的过度积累,现在被描述为泛素化PCNA的新读者,在停滞的叉子被阻止,有助于防止停滞的叉子倒塌。我们进一步确定,ATR通过限制Rad18介导的PCNA单尿素化和端粒上过度的SLX4积累,在端粒(ALT)细胞的替代延长中保持端粒稳定性。这些发现揭示了ATR激活之间复杂的相互作用,Rad18依赖的PCNA单数化,和SLX4相关的停滞叉处理,强调ATR在保持复制叉稳定性和促进端粒酶非依赖性端粒维持方面的关键作用。
Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.