The specific aims of this grant are directed towards a somatic cell genetic analysis of the Ku86:KARP-1 locus in human cells. In the mouse, Ku86, along with Ku70, the DNA-dependent protein kinase catalytic subunit (DNA-PK[CS]), XRCC-4, DNA ligase IV and Artemis, are required for a critical form of DNA double strand break (DSB) repair known as nonhomologous end joining (NHEJ). Recently, we have shown that Ku86:KARP-1 plays an additional, essential role in human somatic cells. In this grant application we provide preliminary data to demonstrate that this essential role is probably related to the ability of Ku86:KARP-1 to regulate telomere length and genomic stability. This is the first demonstration that one of the NHEJ factors regulates these two processes in human cells. We describe below experiments that elucidate the genetic and molecular role(s) of Ku86 and KARP-1 in DNA DSB repair, telomere length regulation and genomic stability in human cells. The importance of identifying and understanding the genes that control human DNA repair is underscored by the existence of a large number of cancer predisposition syndromes such as ataxia telangiectasia, ataxia telangiectasia-like disorder, Nijmegen Breakage syndrome, Fanconi's anemia, Li-Fraumeni syndrome, xeroderma pigmentosum and breast and colon cancer where it appears that the underlying molecular defects reside in DNA repair genes. We intend to characterize these important pathways using four lines of experimentation: 1. Construction of conditional human Ku86:KARP-1-null cell lines. 2. Why do human Ku86:KARP-1-null tissue culture human cells undergo apoptosis? 3. Is the loss of DNA-PK activity responsible for the Ku86:KARP-1-null phenotypes? 4. Is telomerase biogenesis or function aberrant in Ku86:KARP-1 mutant cells? The ultimate goal of these studies is to use Ku86 and KARP-1 mutant cell lines as tools to understand the molecular mechanisms of DNA DSB repair in humans.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Radiation Therapeutics and Biology Study Section (RTB)
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Portnoy, Matthew
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University of Minnesota Twin Cities
Schools of Medicine
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Fattah, Farjana; Lee, Eu Han; Weisensel, Natalie et al. (2010) Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells. PLoS Genet 6:e1000855
Wang, Yongbao; Ghosh, Goutam; Hendrickson, Eric A (2009) Ku86 represses lethal telomere deletion events in human somatic cells. Proc Natl Acad Sci U S A 106:12430-5
Ruis, Brian L; Fattah, Kazi R; Hendrickson, Eric A (2008) The catalytic subunit of DNA-dependent protein kinase regulates proliferation, telomere length, and genomic stability in human somatic cells. Mol Cell Biol 28:6182-95
Fattah, Farjana J; Lichter, Natalie F; Fattah, Kazi R et al. (2008) Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells. Proc Natl Acad Sci U S A 105:8703-8
Fattah, Kazi R; Ruis, Brian L; Hendrickson, Eric A (2008) Mutations to Ku reveal differences in human somatic cell lines. DNA Repair (Amst) 7:762-74
Ghosh, Goutam; Li, Gang; Myung, Kyungjae et al. (2007) The lethality of Ku86 (XRCC5) loss-of-function mutations in human cells is independent of p53 (TP53). Radiat Res 167:66-79