Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

Abstract

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg(2+). At 0.5 mM Mg(2+), where only DNA ligase IV is expected to retain activity, low levels of end joining ( approximately 10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg(2+) in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Transient Association of Ku with Nuclear Substrates Characterized Using Fluorescence Photobleaching

“…Based on the mobility of Ku, the Ku-DSB-nuclear matrix complex must have a half-life of Ͻ10 s. However, if other DNA repair enzymes exhibit the same mobility as Ku, then a shortlived complex of enzymes and DNA may be sufficient for repair of the DSB. Evidence of a DNA-PK-dependent pathway for NHEJ that has a half-time of minutes is consistent with this idea (33). Alternatively, rapid replacement of Ku with additional Ku molecules may establish a steady-state assembly that has a lifetime considerably longer than that of the dwell time of individual Ku molecules.…”

“…However, another interpretation of these data is that DSB repair is complete by 30 min. For example, genetic studies have shown that some forms of NHEJ occur with a half-time of minutes, and that this is a DNA-PK CS -dependent pathway (33). To determine whether the failure to detect changes in the mobility of Ku following irradiation was due to rapid repair of DSBs, cells were pretreated with wortmannin before irradiation to inhibit DNA-PK CS -dependent DSB repair.…”

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

“…The cell cycle dependency of NHEJ is even more contested, and in many reviews it is proposed to act only in G1 phase. This is due to the fact that a large majority of NHEJ-defective cells are hypersensitive to IR when irradiated in G1 but not in G2 (Stamato et al, 1988;Jeggo, 1990;Lee et al, 1997;Takata et al, 1998;Wang et al, 2001). Recently, this interpretation found molecular support by data showing that KU80 preferentially binds to DSBs in the G1 phase (Rodrigue et al, 2006).…”

Ku affects the ATM-dependent S phase checkpoint following ionizing radiation

“…Ku is a heterodimeric protein, containing two subunits, Ku80 and Ku70, both of which bind with high affinity to double-strand DNA (dsDNA) ends. Ku recruits and activates DNA-dependent protein kinase catalytic subunits (DNA-PKcs), and promotes end ligation catalyzed by several DNA ligases (Nick McElhinny et al, 2000;Ramsden and Gellert, 1998;Teo and Jackson, 2000;Wang et al, 2001). Ku is an essential component of DNA double-strand breaks (DSBs) non-homologous end-joining (NHEJ) repair pathway in mammalian cells, and its deficiency leads to extreme sensitivity to IR (Li et al, 1998;Nussenzweig et al, 1996Nussenzweig et al, , 1997Zhu et al, 1996).…”