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Role of Dnl4–Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nature Structural & Molecular Biology volume 14, pages 639–646 (2007)Cite this article

Abstract

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70–Yku80 (Ku), Dnl4–Lif1 and Mre11–Rad50–Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4–Lif1. Notably, Dnl4–Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4–Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4–Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.

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Figure 1: Interaction of Ku, MRX and Dnl4–Lif1 with DNA visualized by SPR.
Figure 2: DNA–protein complex formation by Ku, Dnl4–Lif1 and MRX in EMSAs.
Figure 3: Assembly of NHEJ factors at an in vivo DSB site in mutant strains lacking one of the core NHEJ proteins.
Figure 4: Genetic inactivation of the Dnl4–Lif1 complex results in increased cellular resistance to DNA-damaging agents that cause DSBs.
Figure 5: Dnl4–Lif1 suppresses mating type gene conversion at G1.
Figure 6: Dnl4–Lif1 suppresses HR by inhibiting end resection and Rad51 filament formation.

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Acknowledgements

We thank J. Haber and members of the S.E.L. and A.E.T. laboratories for comments and discussion of the manuscript and M. Murphy for help with design and interpretation of the SPR experiments. This work was supported by a pilot grant from San Antonio Cancer Institute (to S.E.L.) and the US National Institutes of Health (grants ES012244 to S.E.L., GM47251 to A.E.T. and R01ES07061 to P.S. and a Structural Cell Biology of DNA Repair Program grant CA92584 to A.E.T. and P.S.).

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  1. Yu Zhang, Melissa L Hefferin and Ling Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, Texas, 78245, USA

    Yu Zhang, Eun Yong Shim & Sang Eun Lee

  2. Department of Radiation Oncology and the Marlene and Stewart Greenebaum Cancer Center, Radiation Research Laboratory, University of Maryland School of Medicine, Baltimore, 21201-1509, Maryland, USA

    Melissa L Hefferin, Ling Chen, Hui-Min Tseng & Alan E Tomkinson

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06520-8024, Connecticut, USA

    Youngho Kwon & Patrick Sung

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Contributions

Y.Z contributed the data for Figures 3,4,5,6 and Supplementary Figures 3,4,5. M.L.H. contributed purified Ku and the data for Figure 1 and Supplementary Figure 1. L.C. contributed purified Ku, initial ChIP data, purified Dnl4–Lif1 and the data for Figure 2. E.Y.S. and Y.Z. produced Supplementary Figure 2. H.-M.T. contributed the Lif1 antibody and purified Dnl4–Lif1. Y.K. and P.S. contributed purified MRX. A.E.T and S.E.L. directed the project and analyzed the data. A.E.T., S.E.L. and P.S. wrote the manuscript.

Corresponding authors

Correspondence to Sang Eun Lee or Alan E Tomkinson.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

DNA binding by MRX and Dnl4-Lif1 visualized by SPR (PDF 163 kb)

Supplementary Fig. 2

Kinetics of recruitment of the core NHEJ factors to an in vivo DSB (PDF 231 kb)

Supplementary Fig. 3

Kinetics of DSB induction at the MAT locus in haploid yeast strains with the indicated gene deletions (PDF 134 kb)

Supplementary Fig. 4

The increased binding of Ku protein to a DSB in the absence of a functional MRX complex is not due to inefficiency of end resection (PDF 189 kb)

Supplementary Fig. 5

FACS profiles of G1-arrested JKM161 or SLY1A derivatives (PDF 369 kb)

Supplementary Table 1

Calculated equilibrium dissociation constants using a 1:1 Langmuir fitting model (PDF 44 kb)

Supplementary Table 2

Genotypes of strains used in this work (PDF 71 kb)

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Zhang, Y., Hefferin, M., Chen, L. et al. Role of Dnl4–Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination. Nat Struct Mol Biol 14, 639–646 (2007). https://doi.org/10.1038/nsmb1261

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