ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.

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. Synopsis: The exact molecular interplay between ATR activation and Rad18-dependent PCNA monoubiquitination, along with the broader physiological implications of this interaction, remain unclear. This study reveals that ATR restricts Rad18-mediated PCNA monoubiquitination to maintain replication fork and telomerase-independent telomere stability. ATR phosphorylates human Rad18 at Serine 403 in response to replication stress. ATR restrains excessive SLX4 accumulation at stalled forks by phosphorylating Rad18. SLX4 recognizes monoubiquitinated PCNA to promote fork collapse. ATR preserves telomere stability in ALT cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. Replication stress induced phosphorylation of Rad18 restrains excessive SLX4 nuclease accumulation at stalled forks and ALT telomeres. [ABSTRACT FROM AUTHOR]