WASp modulates RPA function on single-stranded DNA in response to replication stress and DNA damage

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Genomic instability, a hallmark of Wiskott-Aldrich syndrome (WAS), arises from perturbations in the replication-stress response (RSR) and DNA-damage response (DDR).
• The precise mechanisms underlying genomic instability in WAS remain largely uncharacterized.
• Replication protein A (RPA), a critical single-strand DNA (ssDNA) binding protein, plays vital roles in both RSR and DDR.

Purpose of the Study:

• To elucidate the role of Wiskott-Aldrich syndrome protein (WASp) in modulating RPA function at perturbed replication forks.
• To investigate the impact of WASp deficiency on DNA replication and repair pathways.
• To understand the evolutionary conservation of WASp's function in DNA stress response.

Main Methods:

• Investigated the localization and function of WASp at replication forks following genotoxic insult in human lymphocytes.
• Assessed the impact of WASp deficiency on the accumulation of key DDR proteins (RPA, ATR, ETAA1, TOPBP1) and CHK1 activation.
• Utilized yeast (Saccharomyces cerevisiae) models deficient in the WASp homolog (Las17) to study DNA recombination and double-strand break repair dynamics.

Main Results:

• WASp accumulates at replication forks upon genotoxic insult, associates with RPA, and promotes RPA:ssDNA complex formation.
• WASp deficiency leads to destabilized RPA:ssDNA complexes, impaired recruitment of DDR factors to perturbed replication forks, and reduced CHK1 activation.
• Yeast cells lacking Las17 exhibit decreased RPA accumulation, impaired DNA recombination, and increased DNA double-strand break (DSB)-induced single-strand annealing (SSA), resulting in higher DSB frequencies.

Conclusions:

• WASp plays an essential, evolutionarily conserved role in the DNA stress-resolution pathway by regulating RPA function at replication forks.
• WASp deficiency results in RPA dysfunction, leading to global replication fork dysfunction and increased genomic instability.
• These findings reveal a novel mechanism linking WASp to the maintenance of genomic integrity, crucial for understanding Wiskott-Aldrich syndrome pathogenesis.