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Molecular and Cellular Biology, December 2005, p. 10782-10790, Vol. 25, No. 24
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.24.10782-10790.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mutations of the Yku80 C Terminus and Xrs2 FHA Domain Specifically Block Yeast Nonhomologous End Joining

Phillip L. Palmbos, James M. Daley, and Thomas E. Wilson^*

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602

Received 23 July 2005/ Returned for modification 18 August 2005/ Accepted 23 September 2005

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic -helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.

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* Corresponding author. Mailing address: Department of Pathology, University of Michigan Medical School, 1301 Catherine Rd., M4214 Med Sci I, Box 0602, Ann Arbor, MI 48109-0602. Phone: (734) 936-1887. Fax: (734) 763-9476. E-mail: wilsonte{at}umich.edu.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, December 2005, p. 10782-10790, Vol. 25, No. 24
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.24.10782-10790.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Palmbos, P. L., Wu, D., Daley, J. M., Wilson, T. E. (2008). Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 Complex to a Double-Strand Break Requires Interactions With Yku80 and the Xrs2 FHA Domain. Genetics 180: 1809-1819 [Abstract] [Full Text]  
• Matsuzaki, K., Shinohara, A., Shinohara, M. (2008). Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1. Genetics 179: 213-225 [Abstract] [Full Text]  
• Wu, D., Topper, L. M., Wilson, T. E. (2008). Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae. Genetics 178: 1237-1249 [Abstract] [Full Text]