Proc Natl Acad Sci U S A 99(2): 832-837

Ku86 is essential in human somatic cells

^{Department of Molecular Biology, Cellular Biology, and Biochemistry, Brown University, Providence, RI 02912; and}^{Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455}

^{Present address: Columbia University, School of Medicine, New York, NY 10027.}

^{To whom reprint requests should be addressed. E-mail:}ude.nmu.ct@460rdneh.

Communicated by Arthur Landy, Brown University, Providence, RI

Received 2001 Oct 5; Accepted 2001 Dec 5.

Abstract

Ku86 plays a key role in nonhomologous end joining in mammals. Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability. In contrast, no human patient has been described with mutations in either Ku86 or Ku70. This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans. To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus. Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation. Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time. These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis. These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.

Abstract

The maintenance of chromosomal integrity is essential for cellular survival (1). Among the many forms of damage that can cause chromosomal instability, DNA double-strand breaks (DSBs) seem to be the most insidious. Improper repair of DSBs results in chromosomal translocations, inversions, and fusions; this, in turn, invariably results in cancer or cell death (1). DSBs can arise through exposure to chemotherapeutic agents or ionizing radiation (IR), occur spontaneously during DNA replication, and are formed transiently in meiosis and during V(D)J recombination in the immune system (1). Cells have evolved at least two independent pathways for repairing DSBs, homologous recombination and nonhomologous DNA end joining (NHEJ; refs. 1 and 2). Homologous recombination ensures accurate repair by using an undamaged sister chromatid or homologous chromosome as a template. NHEJ, on the other hand, uses no, or limited, sequence homology to rejoin ends in a manner that is often error prone. In mammalian cells, NHEJ is the preferred mechanism of DSB repair (2). Some of the gene products involved in this pathway include Ku70, Ku86, the DNA-dependent protein kinase catalytic subunit (DNA–PK_cs), XRCC4, and DNA ligase IV (2).

Ku is a heterodimeric DNA end-binding complex composed of 70- and 86-kDa subunits (Ku70 and Ku86, respectively; ref. 3). Ku binds in a sequence nonspecific fashion to virtually all double-stranded DNA ends including 5′ and 3′ overhangs, blunt ends, and duplex DNA ending in stem-loop structures (3). One unequivocal role for Ku is as a DNA-binding subunit of the DNA-dependent protein kinase (DNA–PK) complex, which is composed of the Ku heterodimer and DNA–PK_cs (3). Extensive genetic and molecular studies have identified the DNA–PK complex as an integral component of mammalian DNA NHEJ DSB repair (3). Ku is believed to bind to broken DNA ends to prevent unnecessary DNA degradation (4) and juxtapose DNA ends (5–7). The binding of Ku to free DNA ends also recruits and activates DNA–PK_cs (8), DNA ligase IV (9, 10), and XRCC4, a DNA ligase IV accessory factor (11, 12), which are required for the rejoining of DNA DSBs (13–16).

Murine knockouts for each of the components of the DNA–PK and XRCC4/ligase IV complexes have been generated. Mice deficient for XRCC4 (12) and DNA ligase IV (17, 18) are not viable because of neuronal degeneration caused by p53-induced apoptosis (19, 20). Mice deficient for Ku70 (21, 22), Ku86 (23, 24), or DNA–PK_cs (25–28) are viable and exhibit the expected immune deficiency and IR hypersensitivity. In addition, inactivation of the Ku86 gene results in cells with growth retardation (23), premature senescence (29), a marked increase in chromosomal aberrations (30–32), and elevated telomeric fusions (33–35).

Whereas DNA ligase IV is an essential gene in rodents, human somatic cells lacking DNA ligase IV are viable (15), and mutations in DNA ligase IV have been described in patients with clinical radiosensitivity and abnormal V(D)J recombination (36, 37). Moreover, functional inactivation in rodents of all three components of the DNA–PK complex has been achieved, yet no human patient has been described with a mutation in any of the subunits. These observations imply that there may be important differences in NHEJ between rodents and humans, and they further suggest that the genes making up the DNA–PK complex may be essential in humans.

To clarify the role of Ku86 in human cells, we have used gene targeting in human somatic tissue culture cells to functionally inactivate the Ku86 locus. Human Ku86 heterozygous cell lines displayed significant haploinsufficient phenotypes; they were defective in cell proliferation and DNA–PK and DNA end-binding activities, and they showed elevated levels of p53, polyploidy, and IR sensitivity. A second round of gene targeting generated homozygously null Ku86 cell lines. These cell lines showed a severe growth defect and ultimately underwent apoptosis after a limited number of cell doublings. These experiments demonstrate that the Ku86 locus is essential in human somatic cells.

Acknowledgments

We thank Dr. J. Sedivy (Brown University) for the gene-targeting construct and for advice on gene targeting early in the conception of this project. We thank Dr. K. Myung (University of California, San Diego) for his efforts in helping to assemble the Ku86 targeting vector. We thank Dr. A. Bielinsky (University of Minnesota) for her helpful comments on the manuscript. This work was supported in part by National Institutes of Health Grant AI35763.

Acknowledgments

Abbreviations

NHEJ	nonhomologous DNA end joining
PK	protein kinase
DSB	double-strand break
IR	ionizing radiation
DT	diphtheria toxin
DEB	DNA end-binding

Abbreviations

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