Probing the functions of mammalian Cdk2 in the DNA damage response with chemical genetics
Final Report Abstract
There is increasing evidence, that mammalian Cdk2, which forms complexes with cyclins E and A functions not only in the regulation of cell cycle progression, but has also a role in promoting DSB repair by HR. Using a chemical genetic approach we were able to prove that acute, specific inhibition of Cdk2 in a non cancer derived human cell line increases its sensitivity to X-ray treatment, indicating a role for Cdk2 in DNA damage response and/or DNA repair. To identify targets of Cdk2 within the DNA damage repair that might be mediators of such an effect, we performed comparative labeling, with the bulky ATP analog N6-(benzyl)-ATP and an analogselective mutant Cdk2/cyclin A complex, in whole-cell extracts of cancer-derived and non-cancer-derived human cells. By this method we identified the Nijmegen breakage syndrome (NBS) gene product Nbs1 as a Cdk2 substrate and mapped the site of phosphorylation to Ser432. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex, which is a key player in DNA double strand break repair. Nbs1-Ser432 is phosphorylated during DNA synthesis (S) and G2 phases in vivo and this phosphorylation is dependent on CDK activity. Nbs1-Ser432 phosphorylation seems to be dispensable for proper checkpoint signaling in response to X-ray irradiation and does not influence general DNA damage sensitivity. It remains to be determined whether this phosphorylation plays some role in regulating the choice of DSB repair pathway. In conclusion, we found that Cdk2 has a function in response to DNA damage caused by DSBs and could identify one possible mediator of this function: Nbs1.
Publications
- Chemical genetics identifies the MRN complex, active in multiple DNA damage response pathways, as a Cdk2 substrate in human cells. June 30th 2008; Genome Integrity Discussion Group meeting, New York Academy of Science
Wohlbold L, De S, Allen JJ, Zhang C, Shokat K, Petrini JHJ and Fisher RP
- Chemical genetics identifies the MRN complex, active in multiple DNA damage response pathways, as a Cdk2 substrate in human cells. The Cell Cycle meeting 2008, Cold Spring Harbor Laboratories
Wohlbold L, De S, Allen JJ, Zhang C, Shokat K, Petrini JHJ and Fisher RP
