title: Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain.
abstract: Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70-Yku80 (Ku), Dnl4-Lif1-Nej1 (DNA ligase IV), and Mre11-Rad50-Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2-Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2-Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80-Dnl4 and Xrs2-Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs-Lif1 and Yku80-Dnl4 interactions are important for formation of a productive ligase-DSB intermediate.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Phillip L
authors.initials: PL
authors.lastname: Palmbos
authors.forename: Dongliang
authors.initials: D
authors.lastname: Wu
authors.forename: James M
authors.initials: JM
authors.lastname: Daley
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C000607891:DNL4 protein, S cerevisiae; C114091:LIF1 protein, S cerevisiae; C000607893:LIG4 protein, human; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; C520770:YKU80 protein, S cerevisiae; D011088:DNA Ligases; D000072481:DNA Ligase ATP