Fission yeast nascent polypeptide-associated complex binds to four-way DNA junctions

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 α and β 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.