A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining.
A predominant pathway implicated in repair of DNA double-strand breaks (DSBs) is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1p, for which a biochemical function has yet to be determined. In this work we demonstrate that Nej1p exhibits a DNA binding activity (KD approximately 1.8 microM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short approximately 20 bp substrates can suffice. This DNA binding activity is the first biochemical evidence supporting the idea that Nej1p plays a direct role in the repair of double-strand breaks. The C-terminus of Nej1p is required for interaction with Lif1p and is sufficient for DNA binding. Structural characterization reveals that Nej1p exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Nej1p (aa 1-244) is shown to be defective in end-joining in vivo. Preliminary functional and structural studies on the Nej1p-Lif1p complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1p and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1p, XLF/Cernunnos.
Sulek
M
M
Department of Biochemistry and Biomedical Sciences, HSC 4N59, McMaster University, 1200 Main Street West, Hamilton, Ont. L8N 3Z5, Canada.
Yarrington
R
R
McGibbon
G
G
Boeke
J D
JD
Junop
M
M
D002850:Chromatography, Gel; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D012996:Solutions
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C114091:LIF1 protein, S cerevisiae; C441270:NEJ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions