Catalytic residues of gamma delta resolvase act in cis.
The resolvase protein of the gamma delta transposon is a site-specific recombinase that acts by a concerted break-and-join mechanism. To analyse the role of individual resolvase subunits in DNA strand cleavage, we have directed the binding of catalytic mutants to specific recombination crossover sites or half-sites. Our results demonstrate that the resolvase subunit bound at the half-site proximal to each scissile phosphodiester bond provides the Ser10 nucleophile and Arg8, Arg68 and Arg71 residues essential for cleavage and covalent attachment to the DNA. Several other residues near the presumptive active site are also shown to act in cis. Double-strand cleavage at one crossover site can proceed independently of cleavage at the other site, although interactions between the resolvase dimers bound at the two crossover sites remain essential. An appropriately oriented heterodimer of active and inactive protomers can in most cases mediate either a 'top' or 'bottom' single-strand cleavage, suggesting that there is no obligatory order of strand cleavages. Top-strand cleavage is associated with the topoisomerase I activity of resolvase, suggesting that a functional asymmetry may be imposed on the crossover site by the structure of the active synapse.
Boocock
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MR
Department of Molecular Biophysics and Biochemistry, Yale University, Bass Center for Molecular and Structural Biology, New Haven, CT 06520-8114, USA.
Zhu
X
X
Grindley
N D
ND
D001483:Base Sequence; D001665:Binding Sites; D003434:Crossing Over, Genetic; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004251:DNA Transposable Elements; D005816:Genetic Complementation Test; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019895:Transposases
D004251:DNA Transposable Elements; D004254:DNA Nucleotidyltransferases; D019895:Transposases; D004264:DNA Topoisomerases, Type I