ATR protects ongoing and newly assembled DNA replication forks through distinct mechanisms

The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11-and EXO1-mediated nascent DNA degradation from PrimPol-gener-ated, single-stranded DNA (ssDNA) gaps. ATRi also exposes template ssDNA through fork uncoupling and nascent DNA degradation. Electron microscopy reveals that ATRi reduces reversed forks by increasing gap-dependent nascent DNA degradation. At new forks, ATRi triggers MRE11-and CtIP-initiated template DNA degradation by EXO1, exposing nascent ssDNA. Upon PARP inhibition, ATRi preferentially exacerbates gap-dependent nascent DNA degradation at ongoing forks in BRCA1/2-deficient cells and disrupts the restored gap protection in BRCA1-deficient, PARP-inhibitor-resistant cells. Thus, ATR protects ongoing and new forks through distinct mechanisms, providing an extended view of ATR's functions in stabilizing replication forks.

Automated summary

The ATR kinase protects genomic integrity during S phase, and it employs distinct mechanisms to safeguard ongoing and new replication forks. ATR inhibitor enhances nascent DNA degradation at ongoing forks by MRE11 and EXO1, as well as exposes template ssDNA through fork uncoupling. It also triggers template DNA degradation by MRE11, CtIP, and EXO1 at new forks, leading to the exposure of nascent ssDNA.