A role for the human Rad9 -Rad1 -Hus1 DNA clamp complex in DNA repair.

摘要

The human proteins Rad9, Rad1, and Hus1 assemble to form a DNA clamp known as the 9-1-1 complex. The 9-1-1 complex plays an essential role in DNA damage recognition and in cell cycle checkpoint signaling. 9-1-1 is structurally similar to proliferating cell nuclear antigen (PCNA), which stimulates many DNA replication and repair enzymes. Work explained herein shows that the 9-1-1 complex stimulates DNA repair enzymes in addition to its role in damage recognition and checkpoint signaling.;Using purified human proteins we showed that 9-1-1 stimulates flap endonuclease 1 (FEN1), an endonuclease responsible for removing single stranded DNA flaps that arise during DNA replication and repair processes. FEN1 stimulation by 9-1-1 was observed on all flap lengths and configurations tested. We also showed that FEN1 and Rad1 coimmunoprecipitate. Consistent with the hypothesis that 9-1-1 is operative in DNA repair pathways, but not necessarily DNA replication, 9-1-1 does not stimulate the replicative DNA polymerase δ.;We employed a human cell model to examine the effect of Rad9 knockdown on cellular phenotype. We showed that cells depleted of Rad9 exhibit decreased survival following ionizing radiation (IR) and that they require more time to exit IR-induced G2 phase arrest. Using the comet assay, we showed that Rad9 deficient cells also require more time to repair IR-induced DNA strand breaks. These results show that the 9-1-1 complex participates directly in DNA repair in addition to its role in DNA damage sensing.;The 9-1-1 complex stimulates many of the enzymes involved in long patch base excision repair (LP-BER) including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β, FEN1, and DNA ligase I (ligase). However, these stimulatory interactions were studied in minimal enzyme systems. We reconstituted the entire LP-BER reaction in vitro to characterize the link between 9-1-1 and LP-BER. Our results reveal that when FEN1 or ligase are limiting, 9-1-1 can restore repair capacity, but not to the levels expected given 9-1-1 stimulation of FEN1 or ligase in isolation. We offer evidence that pol β stimulates FEN1 activity thereby decreasing the need for 9-1-1 to stimulate FEN1 in the LP-BER system.