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hRad9 Rapidly Binds DNA Containing Double-Strand Breaks and Is Required for Damage-dependent Topoisomerase IIß Binding Protein 1 Focus Formation¹

Division of Cancer Biology and Genetics [D. A. G, B. D. A. B., K. B. K., S. D.], and Departments of Pathology [D. A. G., S. D.], Biochemistry [B. D. A. B., S. D.], and Oncology [S. D.], Queen’s University Cancer Research Institute, Kingston, Ontario, K7L 3N6 Canada

Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. We show here that, when DNA double-strand breaks (DSBs) are specifically generated in a subnuclear area, hRad9 is rapidly retained at the damaged DNA, within 2 min of damage induction. Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/H2AX colocalization still occurs in ATM^-/- fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase IIß binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to H2AX, ablating TopBP1 but not H2AX foci formation. The loss of TopBP1 containing foci, but not of H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1.

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