Abstract
The recombinase of the Salmonella inversion system, Hin, mediates site-specific recombination between two 26 base pairs (bp) inverted repeat sequences (hixL and hixR) which flank a 993-bp DNA segment. We have investigated Hin recognition of, and association with, the hix recombination sites. Nuclease and chemical protection studies with linear and supercoiled DNA substrates demonstrate that Hin initially binds hixL and hixR independently of binding of the other protein components of the inversion system, Fis and HU. DNA-binding assays with mutant recombination sites and methylation interference experiments indicate that the critical bases for Hin recognition of its DNA-binding site are within an 8-bp sequence covering adjacent major and minor grooves of the DNA helix in each of the 12-bp half-sites of the hix recombination sites. The nature of the Hin-hix complexes in these binding studies and the results of gel filtration assays with purified Hin suggests that Hin binds the recombination sites as a dimer. The implications of the nature of the interactions of Hin with its recombination sites on the mechanism of the recombination reaction and on the novel features of DNA recognition by Hin are discussed.
Highlights
Hin, mediates site-specific recombinationbetween two ture of Hin, Fis, and HU interactionws ith the DNA substrate
This consensus sequence consists of two imperfect 12-bp inverted repeats separated by a 2-bp core (Fig. 1B).Nuclease and chemical protection studies with Hin, Gin, and Cin have shown that critical bases for Hin recognition of its DNA-binding the recombinase binding site overlaps this 26-bp consensus sitearewithin an 8-bp sequence covering adjacent sequence in the recombination sites [8,9,10,11,12]
The implications of the nature of the interac- It has been demonstrated that the host factor Fis specifitions of Hin with its recombination sites on the mech- cally interacts with the recombinational enhancer sequences anism of the recombination reaction and on the novel of the Hin, Gin, and Cin inversion systems [10, 11, 14]
Summary
The properties of the Fis-enhancer interactions have been incorporated into a model for the role of the recombinationalenhancer in Hin-mediated site-specific inversion [14,15,16]. Present address: Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892. UCLA School of Medicine) was derived from pMS614 by cleavage at theNcoI site (createdby the A +T transversion at the 2-bp core sequence of h i d ) , filling in with T4 DNA polymerase and dNTPs, andblunt end ligation with T4 DNA ligase (Bethesda Research Laboratory); sequence analysis [17] revealed that 1 bpwas lost, resulting in a 3-bp insertion at the center of h i d. Plasmids pAG410 and pAG415 were constructed as follows. pBR322 wascleaved with SalI, the protuding ends filled in with the large fragment of E. coli DNA polymerase I (Klenow; Bethesda Research Laboratory) and dNTPs, and ligated in the presence of a 500-fold
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