XLF and H2AX function in series to promote replication fork stability.

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair Mechanisms

Background:

• XRCC4-like factor (XLF) is known as a DNA double-strand break repair protein involved in non-homologous end joining (NHEJ).
• Phenotypes observed in XLF-deficient individuals and mice suggest roles beyond NHEJ.
• Understanding XLF's full function is crucial for comprehending genome integrity maintenance.

Purpose of the Study:

• To investigate the function of XLF during DNA replication.
• To elucidate the molecular mechanisms underlying XLF's role at replication forks.
• To determine the interplay between XLF, H2AX, and ATR in response to replication stress.

Main Methods:

• Cell cycle analysis to assess XLF phosphorylation.
• Co-immunoprecipitation to study XLF association with replication factors.
• Replication fork progression and reversal assays.
• Analysis of DNA damage response pathways (e.g., ATR, MRE11) in XLF and H2AX deficient cells.

Main Results:

• XLF is phosphorylated in a cell division cycle 7-dependent manner and associates with the replication factor C complex at replication forks.
• XLF deficiency results in impaired replication fork progression and increased fork reversal.
• Combined loss of XLF and H2AX necessitates ATR activation to prevent DNA loss and triggers a DNA damage response, with H2AX loss conferring ATR dependence for survival.

Conclusions:

• XLF functions in DNA replication, impacting fork stability and progression.
• XLF and H2AX act sequentially to mitigate replication stress caused by MRE11-mediated resection of reversed replication forks.
• These findings reveal a novel role for XLF in safeguarding genome integrity during DNA replication.