Identification of three aspartic acid residues essential for catalysis by the RusA holliday junction resolvase.
RusA is a Holliday junction resolvase encoded by the cryptic prophage DLP12 of Escherichia coli K-12 that can be activated to promote homologous recombination and DNA repair in resolution-deficient mutants lacking the RuvABC proteins. Database searches with the 120 amino acid residue RusA sequence identified 11 homologues from diverse species, including one from the extreme thermophile Aquifex aeolicus, which suggests that RusA may be of ancient bacterial ancestry. A multiple alignment of these sequences revealed seven conserved or invariant acidic residues in the C-terminal half of the E. coli protein. By making site-directed mutations at these positions and analysing the ability of the mutant proteins to promote DNA repair in vivo and to resolve junctions in vitro, we identified three aspartic acid residues (D70, D72 and D91) that are essential for catalysis and that provide the first insight into the active-site mechanism of junction resolution by RusA. Substitution of any one of these three residues with asparagine reduces resolution activity >80-fold. The mutant proteins retain the ability to bind junction DNA regardless of the DNA sequence or of the mobility of the crossover. They interfere with the function of the RuvABC proteins in vivo, when expressed from a multicopy plasmid, an effect that is reproducible in vitro and that reflects the fact that the RusA proteins have a higher affinity for junction DNA in the presence of Mg2+ than do the RuvA and RuvC proteins. The D70N protein has a greater affinity for junctions in Mg2+ than does the wild-type, which indicates that the negatively charged carboxyl group of the aspartate residue plays a critical role at the active site of RusA. Electrostatic repulsions between D70, D72 and D91 may help to form a classical Mg2+-binding pocket.
Bolt
E L
EL
Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Sharples
G J
GJ
Lloyd
R G
RG
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000502:physiology*; D001426:Bacterial Proteins / Q000502:physiology; D002384:Catalysis; D002413:Cations, Divalent; D003090:Coliphages / Q000201:enzymology; D004265:DNA Helicases; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000502:physiology; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects / Q000821:virology; D029968:Escherichia coli Proteins; D045565:Holliday Junction Resolvases; D008969:Molecular Sequence Data; D011836:Radiation Tolerance; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism