A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining

M. Sulek, R. Yarrington, G. McGibbon, J. D. Boeke, M. Junop

    Research output: Contribution to journalArticle

    Abstract

    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 ∼ 1.8 μM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short ∼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.

    Original languageEnglish (US)
    Pages (from-to)1805-1818
    Number of pages14
    JournalDNA Repair
    Volume6
    Issue number12
    DOIs
    StatePublished - Dec 1 2007

    Keywords

    • DNA binding
    • DNA repair
    • Double-strand breaks
    • Lif1p
    • Nej1p
    • Non-homologous end-joining
    • XLF/Cernunnos
    • XRCC4

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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