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

Cell Cycle. 2010 Jul 15;9(14):2843-55. doi: 10.4161/cc.9.14.12354. Epub 2010 Jul 11.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Cycle Proteins / metabolism
  • Checkpoint Kinase 1
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • Etoposide / pharmacology
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Mice
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / analysis*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • RNA Interference
  • RNA, Small Interfering / metabolism

Substances

  • Cell Cycle Proteins
  • Histones
  • Mcl1 protein, mouse
  • Myeloid Cell Leukemia Sequence 1 Protein
  • NBN protein, human
  • Nuclear Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Small Interfering
  • Etoposide
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Chek1 protein, mouse