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Xer site-specific recombination in vitro

L K Arciszewska1, D J Sherratt

  • 1Department of Biochemistry, University of Oxford, UK.

The EMBO Journal
|May 1, 1995
PubMed
Summary

XerC and XerD recombinases are crucial for Xer site-specific recombination. An in vitro system shows XerC specifically catalyzes one pair of strand exchanges, clarifying its role in the reaction mechanism.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Genetics

Background:

  • XerC and XerD are lambda integrase family recombinases essential for Xer site-specific recombination in vivo.
  • Understanding their precise roles in the reaction mechanism requires detailed biochemical analysis.

Purpose of the Study:

  • To elucidate the roles of XerC and XerD in the Xer site-specific recombination mechanism.
  • To characterize the catalytic activity and cleavage site of XerC in vitro.

Main Methods:

  • Development of an in vitro recombination system using a synthetic Holliday junction substrate.
  • Utilizing mutant and fusion protein derivatives of XerC and XerD.
  • Characterization of DNA cleavage and strand exchange intermediates.

Main Results:

  • An efficient in vitro recombination system requiring both XerC and XerD was established.
  • Only one specific pair of DNA strands was exchanged, converting the Holliday junction back to the substrate.
  • Mutational analysis identified XerC as the catalyst for this strand exchange.
  • The XerC cleavage site was localized between the XerC binding site and the central region, forming a covalent XerC-DNA complex.

Conclusions:

  • XerC is the primary catalyst for a specific strand exchange event in Xer recombination.
  • The study precisely located the XerC cleavage site and identified a covalent intermediate.
  • This work provides critical insights into the Xer recombination pathway.

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