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Xer site‐specific recombination in vitro.
Author(s) -
Arciszewska L.K.,
Sherratt D.J.
Publication year - 1995
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1995.tb07203.x
Subject(s) - holliday junction , recombinase , site specific recombination , biology , integrases , recombination , dna , flp frt recombination , binding site , nucleotide , cleavage (geology) , integrase , biophysics , genetics , homologous recombination , genetic recombination , gene , paleontology , fracture (geology)
Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site‐specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction‐containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose‐binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC‐mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5′‐OH and a covalent complex between XerC and the 3′ end of the DNA.