Proc Natl Acad Sci U S A 95(24): 14066-14070

DNA end-joining catalyzed by human cell-free extracts

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, United Kingdom

^{Present address: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215.}

^{To whom reprint requests should be addressed.}

Communicated by Martin Gellert, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD

Received 1998 Aug 14; Accepted 1998 Oct 2.

Abstract

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PK_cs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.

Keywords: DNA repair/recombination/Ku protein/DNA-PK/DNA ligase

Abstract

Double-strand breaks are caused, directly or indirectly, by a variety of DNA damaging agents, including ionizing irradiation. To restore the integrity of the genome, cells repair these breaks by (i) homologous recombination, in which an intact chromosome serves as a template for repair, or (ii) nonhomologous end-joining (NHEJ). The latter pathway can be accurate, involving no sequence losses or additions at the joint, or may be inaccurate, a consequence of nucleolytic processing before religation. In simple eukaryotes, such as yeast, homologous recombination provides the dominant pathway for double-strand break repair. Mammalian cells, however, appear to repair the majority of breaks by homology-independent mechanisms.

Mutations in the XRCC4, XRCC5, XRCC6, and XRCC7 genes cause defects in NHEJ resulting in a sensitivity to ionizing radiation and immunodeficiency because of an inability to complete V(D)J recombination (1). XRCC4 encodes a 38-kDa protein that associates with DNA ligase IV and stimulates its ligase activity (2–4), XRCC5 and XRCC6 encode the 86- and 70-kDa subunits of Ku protein (5–7), and XRCC7 encodes the 460-kDa PI 3-like kinase DNA-PK_cs (8, 9). DNA-PK_cs is activated by association with the Ku heterodimer, which exhibits a high affinity for DNA ends (10–13). In Saccharomyces cerevisiae, homologues of Ku70 and Ku86 have been shown to play a role in NHEJ and telomere maintenance (14–20).

Mammalian cells contain three distinct joining activities, named DNA ligases I, III, and IV, which differ greatly in their ability to promote end-to-end ligation (21–23). Ku stimulates intermolecular end-ligation, but not nick-ligation, by all mammalian DNA ligases, presumably by providing a “bridge” between DNA ends without blocking them from the ligase (24, 25). Of the three ligases, DNA ligase IV in particular has been implicated in NHEJ because it interacts with the XRCC4 gene product (3, 4), and xrcc4 mutants are deficient in end-joining (2, 26, 27). However, the ability of ligase IV to promote end-joining in the presence of Ku and Xrcc4 is poor in vitro (24, 28). For it to promote NHEJ in vivo, ligase IV must, therefore, require other, as yet unknown factors. Unfortunately, efforts to study NHEJ in cell-free extracts so far have met with little success. Indeed, although several in vitro systems for DNA end-joining have been described (29–32), it has not been possible to show that these reactions depend on the factors implicated by genetic studies.

In this paper, we demonstrate that extracts prepared from human cells promote efficient intermolecular end-joining of complementary and blunt ends. The end-joining reaction is mostly accurate, occurring without nucleotide loss or addition, and is catalyzed by DNA ligase IV/Xrcc4. NHEJ by the human cell-free extract requires all activities known to be involved in cellular resistance to x-irradiation and V(D)J recombination, namely Ku70, Ku86, and DNA-PK_cs. In addition, end-joining by fractions containing DNA-PK, DNA ligase IV, and Xrcc4 was found to be stimulated by other, as yet unidentified factors.

Acknowledgments

We are indebted to the Jackson and Lindahl laboratories for the generous gifts of antibodies and proteins, and to Deborah Barnes, Richard Bowater, Steve Jackson, Tomas Lindahl, Peter Robins, and Graeme Smith for helpful discussions. This work was supported by the Imperial Cancer Research Fund and the Human Frontiers Science Program.

Acknowledgments

ABBREVIATION

NHEJ	nonhomologous end-joining

ABBREVIATION

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