Ku80-deficient Cells Exhibit Excess Degradation of Extrachromosomal DNA

doi:10.1074/jbc.271.24.14405

Ku80-deficient Cells Exhibit Excess Degradation of Extrachromosomal DNA*

Feng Liang and
Maria Jasin^‡

From the Molecular Biology and the Cell Biology and Genetics Programs, Sloan-Kettering Institute and Cornell University Graduate School of Medical Sciences, New York, New York 10021

^‡ To whom correspondence should be addressed:
Cell Biology and Genetics Program, Sloan-Kettering Institute and Cornell University Graduate School of Medical Sciences, 1275 York Ave., New York, NY 10021.
Tel.: 212-639-7438; Fax: 212-717-3317; E-mail: m-jasin{at}mskcc.org

Abstract

Mammalian cells possess a protein complex, termed DNA-PK, which binds to DNA double strand breaks in vitro. The complex consists of the heterodimeric Ku autoantigen and a DNA-dependent protein kinase, DNA-PK_cs. Cell lines that are deficient for components of this complex are sensitive to ionizing radiation and have impaired V(D)J recombination, a site-specific recombination process. We have tested these cell lines for their ability to repair double strand breaks in transfected DNA. The xrs-6 cell line, which is deficient for the 80-kDa subunit of the Ku autoantigen, exhibited reduced stability of transfected DNA. Prior to obvious reductions in DNA stability, the levels of homologous recombination and DNA end joining were unaffected. However, the recovery of end joining products with precisely joined ends was reduced, with a concomitant increase in products containing deletions. Unlike the Ku80-deficient cells, no reduction in DNA stability was detected in DNA-PK_cs-deficient scid cells. Scid cells also exhibited normal levels of homologous recombination and DNA end joining. These experiments implicate the Ku autoantigen, but not DNA-PK_cs, in a direct role in protecting DNA ends from degradation.

Footnotes

↵* This research was supported by Grant MCB-9419507 from the National Science Foundation, the Frederick R. Adler Chair, and the Pew Charitable Trusts. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DNA-PK

DNA-dependent protein kinase

bp

base pair(s)

Tet^R

tetracycline resistance

Amp^R

ampicillin resistance

Kan^R

kanamycin-resistant

CHO

Chinese hamster ovary

kb

kilobase(s)

Kan^S

kanamycin-sensitive.
↵² P. Romanienko and M. Jasin, unpublished results.
- Received February 26, 1996.