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Strand asymmetry in the repair of replication dependent double-strand breaks

Michael T Kimble, Aakanksha Sane, Robert Jd Reid

    Biorxiv : the Preprint Server for Biology
    |July 1, 2024
    PubMed

    Insights

    DNA replication can turn single-strand breaks into double-strand breaks. Budding yeast repair these breaks via homologous recombination, with the replication-coupled nucleosome assembly pathway crucial for leading strand breaks.

    Area of Science:

    • Molecular Biology
    • Genetics
    • DNA Repair

    Background:

    • Single-strand breaks (SSBs) are common DNA lesions.
    • Replication fork collapse at SSBs can lead to double-strand breaks (DSBs), which are cytotoxic.
    • Understanding the repair mechanisms of replication-dependent DSBs is crucial.

    Purpose of the Study:

    • To investigate the mechanism of replication fork collapse at SSBs.
    • To identify factors involved in the repair of replication-dependent DSBs.
    • To elucidate the role of specific pathways in repairing DSBs arising from leading vs. lagging strand nicks.

    Main Methods:

    • Utilized Cas9 nickase (nCas9) to create site- and strand-specific nicks in budding yeast.
    • Performed genome-wide screens to identify genes essential for DSB repair.
    • Employed various gRNAs to target nicks to either the leading or lagging strand template.

    Main Results:

    • nCas9-induced nicks convert to double-ended DSBs during S-phase.
    • Repair of these DSBs relies on homologous recombination (HR) and not canonical non-homologous end joining.
    • The replication-coupled nucleosome assembly (RCNA) pathway is specifically required for repairing DSBs resulting from leading strand template nicks.

    Conclusions:

    • Homologous recombination is the primary repair pathway for replication-dependent DSBs.
    • The RCNA pathway plays a critical role in resolving fork collapse when the leading strand template is compromised.
    • This study reveals distinct requirements for repairing DSBs originating from leading versus lagging strand damage during replication.

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