Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution.
BACKGROUND
Crystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA-binding cleft, whose surface contains eight basic residues.


RESULTS
To elucidate the functional roles of the basic residues on the cleft surface, we constructed a series of mutant ruvC genes and characterized their properties in vivo and in vitro. Among them, two RuvC mutants with a single alteration, K107A and K118A, were defective in UV-repair and showed a dominant negative effect. The purified K107A and K118A proteins showed reduced binding activity to the junction DNA in the presence of Mg2+ under high salt conditions. Mn2+ increased both the junction binding and cleaving activities of the mutant proteins. In the absence of a divalent cation, the wild-type, K107A and K118A proteins did not bind to junction DNA under high salt conditions, but the D7N mutant, with an alteration of the catalytic centre, was able to bind to the junction efficiently.


CONCLUSIONS
The results presented here, in conjunction with previous crystallographic studies, suggest that the catalytic complex which is formed through interactions of acidic residues, Mg2+ and a cleavable phosphodiester bond, is stabilized by Lys-107 and Lys-118 via electrostatic interactions with the DNA backbone, a process which is critically important for the cleavage reaction to take place. One or two basic residues near the catalytic centre have also been found in other RNase H superfamily proteins, indicating that this is the conserved reaction mechanism in this superfamily.
Yoshikawa
M
M
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Iwasaki
H
H
Kinoshita
K
K
Shinagawa
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008239:Lysine / Q000737:chemistry*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D008345:Manganese; D012965:Sodium Chloride; D004706:Endodeoxyribonucleases; D008274:Magnesium; D008239:Lysine