Human LIGIV is synthetically lethal with the loss of Rad54B-dependent recombination and is required for certain chromosome fusion events induced by telomere dysfunction

Abstract

Classic non-homologous end joining (C-NHEJ) is the predominant DNA double-strand break repair pathway in humans. Although seven genes Ku70, Ku86, DNA-PKcs, Artemis, DNA Ligase IV (LIGIV), X-ray cross-complementing group 4 and XRCC4-like factor are required for C-NHEJ, several of them also have ancillary functions. For example, Ku70:Ku86 possesses an essential telomere maintenance activity. In contrast, LIGIV is believed to function exclusively in C-NHEJ. Moreover, a viable LIGIV-null human B-cell line and LIGIV-reduced patient cell lines have been described. Together, these observations suggest that LIGIV (and hence C-NHEJ), albeit important, is nonetheless dispensable, whereas Ku70:Ku86 and telomere maintenance are essential. To confirm this hypothesis, we inactivated LIGIV in the epithelial human cell line, HCT116. The resulting LIGIV-null cell line was viable, verifying that the gene and C-NHEJ are not essential. However, functional inactivation of RAD54B, a key homologous recombination factor, in the LIGIV-null background yielded no viable clones, suggesting that the combined absence of RAD54B/homologous recombination and C-NHEJ is synthetically lethal. Finally, we demonstrate that LIGIV is differentially required for certain chromosome fusion events induced by telomere dysfunction—used for those owing to the overexpression of a dominant negative version of telomere recognition factor 2, but not used for those owing to absence of Ku70:Ku86.

Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

“…We thus picked puromycin-resistant colonies derived from pHPRT2.2-Puro(−)-transfected cells and confirmed that correctly targeted clones are actually obtained (1 out of 127 clones in wild-type cells and 3 out of 45 clones in LIG4 -null cells) (Figure 3A). The increased targeting efficiency associated with LIG4 deficiency is consistent with previous studies using human cells [7], [28]. Of note, although pHPRT2.2-Puro(−) exhibited low targeting efficiencies compared to pHPRT8.9-Puro(-), gene-targeting enhancement associated with the LIG4 deficiency was more pronounced for pHPRT2.2-Puro(−) (∼10-fold increase) than for pHPRT8.9-Puro(−) (∼2–3-fold increase)(Figure 3A).…”

Alternative end-joining pathway(s): Bricolage at DNA breaks

“…From biochemical data based on the rejoining of a linear plasmid mediated by cellular extracts or purified proteins, we have proposed a double lock model against end joining at telomeric ends in which the TRF2/RAP1 complex inhibits C-NHEJ, whereas the DNA-PK complex blocks A-EJ [187]. This model was further substantiated and refined by data obtained in cells [63,84,188]. Several reports have characterized the first lock level, demonstrating that different shelterin components sustain different protective roles.…”

“…Biochemical studies in our lab have demonstrated that TRF2/RAP1 prevents proper Ku binding and DNA-PK activation at telomeric ends [187]. Thus, removal of TRF2 from telomeres provokes end-to-end chromosome fusions [195,196], which are mainly dependent on Lig4 activity [188,195,197,198]. Moreover, these C-NHEJ dependent end-fusions are completely abrogated in an ATM deficient background [190] and require the C-NHEJ facilitator 53BP1 [84].…”

“…Spontaneous chromosome-end fusions are promoted by a deficiency in either Ku [188,[202][203][204][205][206][207] or DNA-PKcs [208,209], while at least in the absence of Ku, the core shelterin complex appears broadly normal [197]. Furthermore, depletion of Ku in TPP1/POT1 depleted cells or in shelterin-free telomeres provokes a dramatic increase of A-EJ dependent chromosome-end fusions [63,84].…”

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

“…To determine the relative contribution of C-(Ku-and LIG4-dependent) or A-(PARP1-and LIG3-mediated) NHEJ repair components to the telomere fusion events resulting from 17p TALEN activity, we performed parallel nucleofections into HCT116 cell lines deficient for individual (Oh et al 2013B Ruis, T Takasugi, S Oh, EA Hendrickson, in prep. ) and combined components (Supplemental Fig.…”