Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution

Journal of Molecular Biology
Erin R Sanders, R C Johnson

Abstract

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.

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Citations

Aug 4, 2005·Nature Structural & Molecular Biology·Phoebe A Rice
Nov 19, 2008·Nucleic Acids Research·Femi J OlorunnijiW Marshall Stark
Jun 12, 2009·Nucleic Acids Research·Gautam DharReid C Johnson
Feb 9, 2007·Proceedings of the National Academy of Sciences of the United States of America·Mohamad A AbbaniRobert T Clubb
Jun 11, 2009·Molecular Microbiology·Sally-J RowlandW Marshall Stark
Oct 19, 2006·Journal of Molecular Biology·Dunja SkokoReid C Johnson
Apr 2, 2016·Scientific Reports·Botao XiaoReid C Johnson
Sep 28, 2010·Journal of Molecular Biology·Kent W MouwPhoebe A Rice
Jan 10, 2018·Nucleic Acids Research·Caitlin S TrejoPhoebe A Rice
Nov 23, 2010·Critical Reviews in Biochemistry and Molecular Biology·Wei Yang
Jun 25, 2015·Microbiology Spectrum·Reid C Johnson

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