Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

Abstract

The tnpR gene of transposon Tn3 encodes a site-specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co-integrate intermediates of interreplicon transposition to the normal transposition end-products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA-binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120-bp region between the tnpA and tnpR genes that can be subdivided into three separate protein-binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120-bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that onedimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

“…In both regions, a central dinucleotide 5Ј-TT-3Ј is flanked by the sequences 5Ј-GGNCNAAANCN-3Ј and 5Ј-CNGTTT-3Ј, respectively, which are parts of inverted repeats. A similar pattern of sequences is found in the res region of Tn2501, a cryptic transposon of the Tn3 family (11,12). The results confirm that integration of DNA into the genome of strains PdX12 and -14 was the result of a second type of integrative recombination differing from the one mediated by IS1248.…”

“…The result of the latter type of integration is different from that observed for IS1248, in that the integrated DNA is not flanked by two identical sequences. Furthermore, the DNA sequences of the donor backbone and the target DNA at the integration site were found to be similar and to resemble the res site found in transposons belonging to the Tn3 family (11,12). These res sites are an essential part of the transposon-mediated sitespecific recombination system involved in cointegrate resolution.…”

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1

“…Recombinases of the resolvase family cut and religate the DNA within a region of perfect dyad symmetry at the center of res subsite 1 (25). The res site typically contains three subsites, each of imperfect dyad symmetry and each believed to bind a dimer of resolvase (12,19). A specific recombination site with three subsites was searched for within the attP region of TP901-1, but no obvious sites were found.…”

Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase.

“…The Tn3 les recombination site consists of three discrete binding sites for resolvase; the specialized subsite for strand exchange (subsite I, the crossover subsite) is adjoined by two accessory binding sites (subsites II and III) that are essential for efficient recombination in vivo and in vitro (see Fig. 5, below;Grindley et al 1982;Kitts et al 1983;Wells and Grindley 1984). Accessory binding sites for the recombinase or additional protein factors are a characteristic feature of other recombination systems with a strmgent topological selectivity (Sadowski 1986).…”

“…The closely related Hin/Gin/Cin DNA invertases exhibit an analogous inversion selectivity. Contrasting models to account for resolution selectivity emphasize processive "tracking" or "scanning" of resolvase along the DNA (Kitts et al 1983;Krasnow and Cozzarelli 1983), "slithering" of plectonemically supercoiled DNA (Benjamin and CozzareUi 1986), and topological restrictions on the formation of an interwrapped synapse (Boocock et al 1986). …”