ATM promotes reversed fork processing during DNA interstrand cross-link repair

Area of Science:

• DNA repair mechanisms
• Cellular response to DNA damage
• Molecular cell biology

Background:

• Replication-coupled DNA interstrand cross-link (ICL) repair involves crucial fork remodeling events.
• The regulation of fork reversal and restoration during ICL repair remains poorly understood.
• The Fanconi anemia (FA) pathway is central to resolving ICLs.

Purpose of the Study:

• To investigate fork dynamics during ICL repair by the FA pathway using cell-free Xenopus egg extracts.
• To elucidate the regulatory mechanisms governing reversed fork processing during ICL repair.

Main Methods:

• Utilized cell-free Xenopus egg extracts to model ICL repair.
• Investigated the role of ataxia telangiectasia-mutated (ATM) kinase in fork reversal.
• Assessed the functions of EXO1 and DNA2 nucleases in reversed fork resection.
• Examined the impact of protein phosphatase 2A (PP2A) inhibition on ATM signaling and fork processing.

Main Results:

• ATM kinase activation is concomitant with fork reversal and promotes resection of the reversed fork intermediate.
• EXO1 and DNA2 nucleases coordinate to resect the reversed fork, with EXO1 initiating and DNA2 performing further resection.
• Inhibition of PP2A leads to ATM hyperactivation, excessive reversed fork resection, and aberrant end-joining products.
• Reversed forks are identified as substrates for ATM activation.

Conclusions:

• A phospho-regulatory circuit involving ATM and PP2A governs reversed fork processing during ICL repair.
• This circuit ensures controlled resection of reversed forks, preventing aberrant DNA repair outcomes.
• The findings provide new insights into the regulation of DNA replication fork dynamics during ICL repair.