CtIP is required to initiate replication-dependent interstrand crosslink repair

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions that impede DNA replication and transcription.
• CtIP is a known DNA repair protein involved in processing double-strand breaks (DSBs) via homologous recombination.
• The specific role of CtIP in ICL repair, particularly in replicating cells, remains to be fully elucidated.

Purpose of the Study:

• To investigate the role of CtIP in the processing of DNA interstrand crosslinks (ICLs) in human cells.
• To determine if CtIP's function in ICL repair is distinct from its established role in DSB repair.
• To elucidate the molecular mechanisms by which CtIP participates in ICL repair initiation.

Main Methods:

• Utilized laser-induced ICLs and DSBs in human cells to study DNA damage response.
• Employed CtIP depletion (e.g., siRNA) to assess its necessity in ICL and DSB repair pathways.
• Monitored the recruitment of key DNA damage response proteins (RPA, ATR, FANCD2, γH2AX, p-ATM) to damage sites using microscopy.

Main Results:

• CtIP depletion significantly sensitizes human cells to ICL-inducing agents.
• CtIP deficiency impairs the accumulation of RPA, ATR, FANCD2, γH2AX, and p-ATM at laser-generated ICL sites.
• In contrast, the recruitment of γH2AX and p-ATM to laser-generated DSBs is independent of CtIP.
• CtIP acts early in ICL repair, dependent on BRCA1 and FANCM, before DSB formation.

Conclusions:

• CtIP plays a critical, previously unrecognized role in the early stages of ICL repair initiation in replicating human cells.
• This function is distinct from CtIP's role in canonical DSB repair, highlighting pathway specificity.
• CtIP acts upstream of DSB formation in ICL processing, in conjunction with BRCA1 and FANCM.