A requirement for polymerized actin in DNA double-strand break repair

Abstract

Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

Keywords: :

• nuclear actin
• DSB repair
• DNA damage
• polymeric actin
• Ku70
• Ku80

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Randall Gieni for critical reading and editing of the manuscript. We would like to thank Dr. Dick van Gent for the kind gift of the V15B (Ku80 deficient) and Ku80-GFP reconstituted CHO cell lines as well as the Ku80-GFP plasmid construct and Dr. Ola Hammarsten for the kind gift of the purified Ku heterodimer. This work was supported by an operating grant from the National Cancer Institute of Canada to MJH and a Discovery Grant from the Natural Science and Engineering Research Council (NSERC) to GD. MJH is a senior scholar of Alberta Innovates Health Solutions and GD is Canadian Institutes of Health Research (CIHR) New Investigator and Senior Scientist of the Beatrice Hunter Cancer Research Institute (BHCRI). KMA was supported by the BHCRI with funds provided by The Terry Fox Foundation Strategic Health Research Training (STIHR) Program in Cancer Research at CIHR and is currently supported by a studentship award from NSERC.