ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links

Abstract

SummaryInterstrand cross-links (ICLs) are toxic DNA lesions interfering with DNA metabolism that are induced by widely used anticancer drugs. They have long been considered absolute roadblocks for replication forks, implicating complex DNA repair processes at stalled or converging replication forks. Recent evidence challenged this view, proposing that single forks traverse ICLs by yet elusive mechanisms. Combining ICL immunolabeling and single-molecule approaches in human cells, we now show that ICL induction leads to global replication fork slowing, involving forks not directly challenged by ICLs. Active fork slowing is linked to rapid recruitment of RAD51 to replicating chromatin and to RAD51/ZRANB3-mediated fork reversal. This global modulation of fork speed and architecture requires ATR activation, promotes single-fork ICL traverse—here, directly visualized by electron microscopy—and prevents chromosomal breakage by untimely ICL processing. We propose that global fork slowing by remodeling provides more time for template repair and promotes bypass of residual lesions, limiting fork-associated processing.

Replication gaps underlie BRCA-deficiency and therapy response

“…Although gaps are a common indicator of replication stress and result from loss of the BRCA-RAD51 pathway, they have been overlooked as the determinant of toxicity in favor of defects in HR and FP (Hashimoto et al, 2010;Henry-Mowatt et al, 2003;Kolinjivadi et al, 2017;Sugimura et al, 2008;Wang et al, 2015;Xu et al, 2018;Zellweger et al, 2015). However, it is unclear how HR and FP participate in therapy response given that drugs used to treat BRCA deficient tumors do not initially cause DNA breaks or stall forks (Huang et al, 2013;Maya-Mendoza et al, 2018;Mutreja et al, 2018). In contrast, replication gaps arising due to loss of the BRCA-RAD51 pathway provides a logical explanation for the initiating lesion that is exacerbated by chemotherapies that further dysregulate replication.…”

“…However, recent findings challenge the fundamental premise that DSBs are the critical lesion for cisplatin sensitivity. Notably, cisplatin does not initially cause replication forks to stall and collapse (Huang et al, 2013;Mutreja et al, 2018). Moreover, recent findings indicate that cisplatin toxicity in triple negative breast cancer is unrelated to loss of DNA repair factors (Heijink et al, 2019).…”

Replication gaps are a cancer vulnerability counteracted by translesion synthesis

“…During stress, fork slowing and remodeling promote the global arrest of DNA replication and maintain genomic stability (Mutreja et al, 2018) (Figure 1A). Thus, TLS interference with fork slowing in response to stress could limit the global arrest of DNA replication.…”

“…Inhibition of the checkpoint kinase ATR also enables replication during stress (Mutreja et al, 2018) ( Figure 2A). Given that ATR inhibition is toxic to cells (Couch et al, 2013), we considered that a key difference between TLS activation and ATR inhibition was ssDNA gap induction.…”

“…The replication stress response is activated in response to DNA lesions or intrinsic replication fork barriers, and it is critical to ensure the accurate transmission of genetic material to daughter cells (Berti and Vindigni, 2016). In response to stress, replication forks slow and remodel into reversed fork structures and this local fork response confers a signal to arrest DNA replication throughout the cell (Mutreja et al, 2018;Neelsen and Lopes, 2015). Cells either undergo replicative senescence or engage in DNA repair or other transactions that restart stalled DNA replication forks.…”