Analysis of the γδ res site : Sites required for site-specific recombination and gene expression

Abstract

The γδ resolvase, product of the transposon's tnpR gene, mediates a site-specific recombination between two copies of γδ directly repeated on the same replicon. The site at which the recombination occurs, res, lies between the tnpA and tnpR genes. Within this same region lie the promoters for expression of tnpA and tnpR and the operator sites through which resolvase regulates the transcription. In order to determine the extent of the res site we have constructed in vitro a series of deletions that terminate within the tnpA- tnpR intergenic region, and have analyzed their effect on site-specific recombination. Our results indicate that a fully functional res site is about 115 base-pairs (bp) and runs from a position 15 bp to the left ( tnpA-proximal) side of the crossover point to 100 bp to the right. This segment corresponds precisely to the region defined by the three resolvase binding sites that we have demonstrated previously. Alterations of the nucleotide sequence around the crossover point indicate the importance of all or part of the central palindrome 5′ T-T-A-T-A-A within which the breakage-reunion reaction takes place. Taken together, our results strengthen our earlier conclusion that resolvase recognizes the 9 bp segment 5′ T-G-T-C-Y-N-N-T-A that occurs (in slightly degenerate form) in each half of the three binding sites. Using the deletions we have confirmed that the tnpA promoter spans the crossover site and have shown that the major tnpR promoter in vivo coincides with resolvase binding site II, although a second promoter for tnpR transcription lies across site I.