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The four-way DNA junction (X-junction) is both a central intermediate of recombination reactions and, in some cases, a controlling element in transcription and the initiation of DNA replication. Many different proteins have been found to bind to X-junctions in a structure-specific manner. In some cases, this ability only reflects the proteins' general predilection for distorted DNAs but in others the interaction is highly specific and usually signifies that the X-junction is the real target for the protein in vivo. Here we identify the Schizosaccharomyces pombe (Sp) nascent polypeptide associated complex (NAC) as a potent binder of X-junction DNA. NAC is highly conserved in eukaryotes and has reported functions in transcription and the targeting of proteins within the cytosol. NAC is composed of alpha and beta subunits. Each SpNAC subunit has the capacity to bind X-junction DNA, but optimal binding depends on a heterodimer of subunits. Competition assays and binding comparisons using a range of different DNA substrates reveal that SpNAC is highly selective for the X-junction structure. By comparative gel electrophoresis we show that the X-junction is held in its open square conformation when bound by SpNAC. Junction binding is inhibited by concentrations of magnesium ions that are sufficient to "stack" the X-junction, suggesting that SpNAC recognises only the open junction structure. Finally, SpNAC can bind to X-junctions that are already bound by a tetramer of the Escherichia coli RuvA protein, indicating that it interacts with only one face of the junction. The possible biological significance of X-junction binding by SpNAC is discussed.

Original publication

DOI

10.1006/jmbi.2000.4407

Type

Journal article

Journal

J Mol Biol

Publication Date

02/03/2001

Volume

306

Pages

703 - 716

Keywords

Amino Acid Sequence, Cell Extracts, Cloning, Molecular, DNA, DNA Helicases, DNA-Binding Proteins, Dimerization, Escherichia coli Proteins, Magnesium, Molecular Chaperones, Molecular Sequence Data, Nuclear Proteins, Nucleic Acid Conformation, Oligodeoxyribonucleotides, Protein Binding, Protein Conformation, Protein Structure, Quaternary, Recombinant Proteins, Recombination, Genetic, Schizosaccharomyces, Substrate Specificity, Trans-Activators, Transcription Factors