A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining
| Autor: | Robert M. Yarrington, Murray S. Junop, G. McGibbon, M. Sulek, Jef D. Boeke |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Saccharomyces cerevisiae Proteins
HMG-box DNA repair Saccharomyces cerevisiae Electrophoretic Mobility Shift Assay Biology Biochemistry chemistry.chemical_compound DNA Fungal Molecular Biology Replication protein A Cell Biology DNA repair protein XRCC4 biology.organism_classification Molecular biology DNA binding site Non-homologous end joining DNA-Binding Proteins Solutions chemistry Chromatography Gel Mutagenesis Site-Directed Electrophoresis Polyacrylamide Gel Dimerization DNA Protein Binding |
| Zdroj: | DNA repair. 6(12) |
| ISSN: | 1568-7864 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect