Three DNA DSB repair pathways have been identified to date in human somatic cells: HR (homologous recombination), C-NHEJ (classic non-homologous end joining) and A-NHEJ (alternative-NHEJ). Which repair pathway gets used appears to be a function of several parameters although cell type and cell cycle phase are chief amongst them and in human bodies, C-NHEJ is the chief repair pathway.Seven core proteins; Ku70, Ku86, DNA-PKcs, Artemis, LIGIV, XRCC4 and XLF are known to be required for C-NHEJ. Mutations of most of these genes result in cells or animals with profound deficits in DNA DSB repair and severe immune deficiencies. The phenotypes resulting from loss of function of LIGIV, were, however rather variable depending upon what model organism was being study. Consequently, it was unclear whether LIGIV is essential for human cell survival or not. To experimentally address this issue, we functionally inactivated the LIGIV gene and showed that LIGIV (and presumably C-NHEJ) is not essential for human somatic cell survival.LIGIII, one of the three mammalian ligases, was recently implicated as possibly being involved in A-NHEJ pathway. Recent studies carried out in mice indicated that LIGIII had an essential function in mitochondria and was likely involved in chromosomal translocations in nucleus. To study similar phenotypes in human somatic cells we generated a conditionally viable LIGIII-null cell line. We then demonstrated that human LIGIII is also essential for mitochondria function but then went on to show that absence of nuclear LIGIII is tolerated with almost no pathological phenotypes. Finally, we have observed that cells lacking the key C-NHEJ factor, Ku86, succumb because of telomere dysfunction. Several laboratories have postulated that the t-circle formation is due to the aberrant activity of HR on telomeric DNA. To experimentally test this model, we functionally inactivated the Rad54B gene -- a major HR component -- in Ku86 conditional null cells. Impressively, a Rad54B deficiency partially rescues the defects of Ku86-null cells: it significantly delays the death of Ku86-null cells and significantly reduces t-circle formation. These results indicate that the telomere dysfunction observed in Ku86-null cell is, at least in part, mediated by HR.
University of Minnesota Ph.D dissertation. December 2011. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Eric A. Hendrickson. 1 computer file (PDF); viii, 176 pages.
Interaction between the three DNA double strand break repair pathways in human cells.
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