Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

Abstract

Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1⁻ fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G₂/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

ACKNOWLEDGMENTS

We thank Juanita Campos-Torres for extensive assistance with the FACS analysis and Anindya Dutta, Sylvia Lee, Boris Reizis, and Tom Wolkow for helpful discussions and comments on the manuscript.

This work was supported by American Cancer Society postdoctoral fellowship PF-98-131-01 and NCI training grant CA 09361-20 (R.S.W.), the Howard Hughes Medical Institute (P.L.), and NIH grant RO1 ES009558 (C.V.).