Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome.
To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb. The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.
Morel-Deville
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Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France. fmorel@biotec.jouy.inra.fr
Ehrlich
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SD
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D002702:Chloramphenicol Resistance / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D016521:Electrophoresis, Gel, Pulsed-Field; D015180:Electrophoresis, Gel, Two-Dimensional; D013293:Enterococcus faecalis / Q000235:genetics; D005838:Genotype; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012093:Replicon / Q000235:genetics; D017384:Sequence Deletion; D013696:Temperature
D004269:DNA, Bacterial