Role of arginine‐43 and arginine‐69 of the Hin recombinase catalytic domain in the binding of Hin to the <i>hix</i> DNA recombination sites

Adams, Carson; Nanassy, Oliver Z.; Johnson, Reid C.; Hughes, Kelly T.

doi:10.1046/j.1365-2958.1997.4141789.x

Cited by 11 publications

(9 citation statements)

References 34 publications

(58 reference statements)

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“…The amino acid residues G38, R43 and R69 are in the N-terminal catalytic domain, and R103 and M109 are in helix E. Furthermore, the amino acid changes of R49 and R69 (see Fig. 2) have been implicated in the binding activity of Hin (Adams et al 1997). Taking these data together, it may be suggested that the unfavorable interactions between the substituted amino acids in the helix E and the neighboring amino acids in the core of the N-terminal domain in¯uence the relative positions of the two E helices at the dimer interface, which subsequently leads to the impairment of DNA binding activity.…”

Section: Conformational Change In the N-terminal Catalytic Domain Andmentioning

confidence: 98%

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Lee

et al. 2001

Mol Gen Genomics

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Previous biochemical assays and a structural model of the protein have indicated that the dimer interface of the Hin recombinase is composed of two alpha-helices. To elucidate the structure and function of the helix, amino acids at the N-terminal end of the helix, where the two helices make their most extensive contact, were randomized, and inversion-incompetent mutants were selected. To investigate why the mutants lost their inversion activities, the DNA binding, hix pairing, invertasome formation, and DNA cleavage activities were assayed using in vivo and in vitro methodologies. The results indicated that the mutants could be divided into four classes based on their DNA binding activity. We propose that the alpha-helices might serve to place a DNA binding motif of Hin in the correct spatial relationship to the minor groove of the recombination site. All the mutants except those that failed to bind DNA were able to perform hix pairing and invertasome formation, suggesting that the dimer interface is not involved in either of these processes. The inversion-incompetent phenotype of the binders was caused by the inability of mutants to perform DNA cleavage. The mutants that showed less binding ability than the wild type nevertheless exhibited a wild-type level of hix pairing activity, because the hix pairing activity overcomes the defect in DNA binding. This phenotype of the mutants that are impaired in DNA binding suggests that the binding domains of Hin may mediate Hin-Hin interaction during hix pairing.

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Section: Conformational Change In the N-terminal Catalytic Domain Andmentioning

confidence: 98%

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Lee

et al. 2001

Mol Gen Genomics

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“…5B, lane 3, below). Unlike other substitutions at this position (Adams et al 1997), S10G has no detrimental effect on hix binding (M. Lainé and R.C. Johnson, unpubl.)…”

Section: Properties Of Hin Mutants Used To Investigate Hin Activationmentioning

confidence: 99%

Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion

Merickel¹,

Haykinson²,

Johnson³

1998

Genes Dev.

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The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile ␤-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating ␤-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein-DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.

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“…Media conditions, concentrations of antibiotics and lactose indicators, transductional crosses, and transformations were as reported previously (1,5,23).…”

Section: Methodsmentioning

confidence: 99%

“…Binding studies were performed as described previously, with the following modifications (1,23). Various amounts of purified Hin protein were used to determine the apparent K d values.…”

Section: Methodsmentioning

confidence: 99%

“…This plasmid is a Fis expression plasmid containing the ColE1 origin of replication. The Hin and Fis wild-type and mutant proteins were overexpressed in the RJ1632 background and purified as described previously (1,24). The coding sequences for the A131V, S70G, D65N, T124I, and A126T Hin mutants were cloned into the pZN38 vector by digesting this vector with ClaI and HindIII to excise the fragment of the wild-type hin gene encoding the C terminus and replacing it with the same fragment from the pKH66-derived hin mutant expression plasmids (23) to yield the pZN42, pZN56, pZN64, pZN66, pZN68, and pZN70 plasmids.…”

Section: Methodsmentioning

confidence: 99%

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Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

Nanassy

Hughes

2001

J Bacteriol

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A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially The Hin recombinase of Salmonella spp., in conjunction with the Fis and HU accessory proteins, catalyzes a reversible sitespecific recombination reaction that results in the alternate expression of flagellin antigens, a process known as flagellar phase variation. The Hin-Fis site-specific recombination reaction provides a model system for the investigation of the molecular mechanism of genetic recombination. The recombination reaction catalyzed by the Hin recombinase takes place between two chromosomal sites, hixL on the left and hixR on the right, that flank an invertible DNA segment (27). The underlying molecular mechanism of recombination has been shown to require three proteins, Hin, Fis, and HU (14), and three DNA sites, hixL, hixR, and a recombinational enhancer element (RE) (8, 13). Hin dimers bound to the hixL and hixR recombination sites are thought to act with Fis dimers bound to two sites within the RE during the inversion reaction. The role of HU is to bend DNA, facilitating the assembly of a complex between Hin and Fis. Immunoelectron microscopy has permitted visualization of the nucleoprotein intermediate, or invertasome, that is responsible for carrying out strand exchange (10). By analogy to the closely related Gin recombinase system described below, Hin-Fis-directed invertasome formation is thought to trap DNA supercoils such that after recombination four negative supercoils are lost, effectively driving the reaction to completion. The isolated invertasome structure included Hin and Fis, which were colocalized in the crosslinked complex (10). Fis aids the binding of Hin to the hixR site in vivo (23) and activates the Hin dimers within the synaptic complex in order to initiate concerted DNA cleavage (7, 21).In the closely related Gin invertase system, two negative nodes are trapped as a result of invertasome formation (16-18). Strand exchange is initiated after cleavage of the DNA by the recombinase within the invertasome, and single righthanded (clockwise) rotation about the helix axis, followed by religation, results in a net loss of four negative s...

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Role of arginine‐43 and arginine‐69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

Cited by 11 publications

References 34 publications

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion

Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

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