End-joining repair of double-strand breaks in Drosophila melanogaster is largely DNA ligase IV independent - PubMed (original) (raw)

End-joining repair of double-strand breaks in Drosophila melanogaster is largely DNA ligase IV independent

Mitch McVey et al. Genetics. 2004 Dec.

Abstract

Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4-independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies.

PubMed Disclaimer

Figures

F<sc>igure</sc> 1.—

Figure 1.—

The Lig4 region and the lig4169 deletion. The four coding exons for LIG4 are shown as boxes; the shaded box represents the BRCT motif, and the stippled box corresponds to the region responsible for binding CG3448, the putative Drosophila XRCC4 homolog. Transcription of the adjacent CG12176 locus occurs in the opposite direction of Lig4. The P{EP}385 element used to create the lig4169 deletion is located 40 bp upstream of Lig4. The lig4169 deletion extends from −40 to +2330 (+1 corresponds to the translation start site).

F<sc>igure</sc> 2.—

Figure 2.—

lig4169 mutants are not sensitive to ionizing radiation. (A) Third-instar larvae from the cross lig4169 /+ × lig4169/Y were irradiated with indicated doses of gamma rays, and survival rates of lig4169/lig4169 homozygous to lig4169/+ heterozygous female progeny, or lig4169/Y to +/Y male progeny, were calculated. Survival rates are given relative to the ratios observed for the untreated controls. (▵) females, (□) males, (⋄) average. (B) Mutation of lig4 and spn-A results in synergistic sensitivity to intermediate doses of X rays. Double-mutant flies were created by crossing lig4169/FM7; spn-A57/TM3 females to lig4169/Y; spn-A93/TM3 males. Third-instar larvae were exposed to indicated doses of gamma rays and relative survival rates were calculated relative to the survival rate of lig4 spn-A heterozygous females at each dose. (⋄) lig4, (□) spn-A, (▵) lig4 spn-A. Heightened sensitivity was observed in the double mutant at doses between 400 and 500 rad.

F<sc>igure</sc> 3.—

Figure 3.—

Model for repair of _P_-element-induced DSBs in Drosophila. Transposase cuts at both ends of the P element, creating a double-strand gap with 3′ overhanging ends that can invade into the unbroken sister chromatid (shaded) and initiate homologous repair by SDSA. Occasionally, for unknown reasons, SDSA aborts and repair of the broken chromosome occurs by end joining (dashed line). In the absence of the strand-exchange protein DmRad51, SDSA cannot initiate and end joining proceeds by alignment of small microhomologies, presumably followed by trimming of overhanging ends, filling in of gaps, and ligation. The majority of this end-joining repair occurs via a LIG4-independent pathway.

Similar articles

Cited by

References

    1. Adams, M. D., M. McVey and J. J. Sekelsky, 2003. Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science 299: 265–267. - PubMed
    1. Barnes, D. E., G. Stamp, I. Rosewell, A. Denzel and T. Lindahl, 1998. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr. Biol. 8: 1395–1398. - PubMed
    1. Beall, E. L., and D. C. Rio, 1997. Drosophila P-element transposase is a novel site-specific endonuclease. Genes Dev. 11: 2137–2151. - PMC - PubMed
    1. Boulton, S. J., and S. P. Jackson, 1996. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res. 24: 4639–4648. - PMC - PubMed
    1. Boulton, S. J., and S. P. Jackson, 1998. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 17: 1819–1828. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources