Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to γH2AX, ablating TopBP1 but not γH2AX foci formation. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase IIβ binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/γH2AX colocalization still occurs in ATM −/− fibroblasts. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (γH2AX) after DNA damage.

Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. 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. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication.

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