MCL-1 localizes to sites of DNA damage and regulates DNA damage response.

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1(-/-) MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G(1)/S checkpoint response, was not significantly different. In addition, appearance of gamma-H2AX was delayed in the Mcl-1(-/-) MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with gamma-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide , followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.