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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.

Pubmed ID: 17765666

Authors

  • Sulek M
  • Yarrington R
  • McGibbon G
  • Boeke JD
  • Junop M

Journal

DNA repair

Publication Data

December 1, 2007

Associated Grants

  • Agency: NIGMS NIH HHS, Id: R01 GM036481

Mesh Terms

  • Chromatography, Gel
  • DNA, Fungal
  • DNA-Binding Proteins
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoretic Mobility Shift Assay
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Saccharomyces cerevisiae Proteins
  • Solutions