The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome.
Holliday junctions are four-way branched DNA structures formed during recombination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitates loading of RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB) and resolution (RuvABC). We investigated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriate substitutions of two key amino acids (Glu55 and Asp56), we altered the charge on the pin and investigated how this affected junction binding and processing. We show that two negative charges on each subunit of the pin are crucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for junction processing. These findings provide the first direct evidence that RuvA has a mechanistic role in branch migration. They also provide insight into the coupling of branch migration and resolution by the RuvABC resolvasome.
Ingleston
S M
SM
Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
Sharples
G J
GJ
Lloyd
R G
RG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D017124:Conserved Sequence; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases