Checkpoint Responses for Protecting Genome Integrity in S-Phase

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DESCRIPTION (provided by applicant): Protection of genome integrity is critical for preventing genetic mutations and chromosomal rearrangements that can lead to cancer, aging-related disorders, neurological, immunological and developmental diseases, infertility, birth defects and many other disorders. DNA damage triggers the checkpoint kinases ATM and ATR to phosphorylate the C-terminus of histone H2AX in chromatin flanking DNA lesions. This phospho-H2AX, known as gamma-H2AX, serves as a signaling and protein docking platform to regulate DNA repair and cell cycle checkpoint activities. While gamma-H2AX has well known roles at DNA double- strand breaks created by ionizing radiation and other clastogens, its role at stalled or damaged replication forks during the DNA synthesis (S)-phase of the cell cycle is enigmatic. We recently discovered that gamma-HAX recruits the genome maintenance factor Brc1 to stalled or damaged replication forks. We also found that Brc1 binding to gamma-H2AX is crucial in the absence of Rqh1, which is the ortholog of human BLM DNA helicase that is mutated in Bloom's Syndrome. In this project we propose to: (1), discover why ¿H2AX is crucial when Rqh1 DNA helicase is defective; (2), define and characterize the genetic deficiencies that create a critical requirement for ¿H2AX and Brc1; (3), assess the functions of the electronegative surface in the BRCT5-6 interdomain linker of Brc1. The impact of these studies will be to significantly improve the understanding of how genome integrity is protected in S-phase.