Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo


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Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5' and 3' Patches

Homologous recombination in Escherichia colicreates patches (non-crossovers) or splices (half crossovers), eachof which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex,with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism ofrecombination. Several models for the mechanism of E. coliRecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3’-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands.

Methodology/Principle Findings
This paper re-examines heteroduplex-patch-strand polarity using phage l and the ldv plasmid as DNA substrates recombined viathe E. coliRecBCD system in vivo. These DNAs are mutant for l recombination functions, including orfand rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis.

This method reveals that heteroduplexes are still found in either 5’ or 3’ DNA strands in approximately equalamounts, even in the absence of orfand rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation.

These results indicate that orfand rapdid not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation.

We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.

Gumbiner-Russo LM, Rosenberg SM (2007)
Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5' and 3' Patches.
PLoS ONE 2(11): e1242. doi:10.1371/journal.pone.0001242
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