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

Background. Homologous recombination in Escherichia colicreates patches (non-crossovers) or splices (half crossovers), each
of 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 of
recombination. Several models for the mechanism of E. coliRecBCD-mediated recombinational double-strand-end (DSE) repair
specify that only the 39-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 59 or 39 DNA strands in approximately equal
amounts, 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. Conclusions/
Significance. 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.
Citation: Gumbiner-Russo LM, Rosenberg SM (2007) Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of
59 and 39 Patches. PLoS ONE 2(11): e1242. doi:10.1371/journal.pone.0001242


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