Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution.
The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction.
Sanders
Erin R
ER
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Johnson
Reid C
RC
D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D023902:Chromosome Pairing; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004278:DNA, Superhelical; D019281:Dimerization; D035021:Factor For Inversion Stimulation Protein; D007700:Kinetics; D008958:Models, Molecular; D011995:Recombination, Genetic; D012475:Salmonella / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D035021:Factor For Inversion Stimulation Protein; D012694:Serine; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase