The RuvABC Holliday Junction Processing System Is Not Required for IS-Mediated Targeted Conservative Cointegrate Formation.

The insertion sequence IS plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS and members of the IS family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the , , or genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. In Gram-negative bacteria, the contribution of IS to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS family found in Gram-positive as well as Gram-negative pathogens.