Nicking-closing activity associated with bacteriophage lambda int gene product.

Abstract

Integrative recombination of bacteriophage X requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation o nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg +, spermidine, and singlestranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination. Bacteriophage X forms lysogens of Escherichia coli by integrating viral DNA into the bacterial chromosome (reviewed in ref. 1). The integration occurs as a result of a reciprocal recombination between specific genetic loci, the viral and bacterial attachment sites (attP and attB, respectively). Integrative recombination readily takes place in a cell-free system (2), and the in vitro reaction has been used to characterize the overall pathway of this recombination. Briefly, it has been shown that DNA supercoiling is essential for recombination: at least attP must be located on a negatively supertwisted circle of DNA (3,4). Given an appropriately supertwisted substrate, the only low molecular weight substances required for full recombination activity are spermidine, KC1, and buffer. The products of integrative recombination are a reciprocal pair of continuous double-helical DNAs. This fact and the simplicity of the cofactor requirements suggest that recombination occurs by a very conservative mechanism-i.e., one that involves-no degradation or resynthesis of DNA and one that retains the bond energy of the phosphodiester backbone during breakage and reunion.In vitro integrative recombination has been used to assay the purification of the proteins involved in this reaction. We have previously reported the purification to near homogeneity of the X int gene product, the only virus-coded component of the integration system (5, 6). We showed that, in addition to supertwisted substrate DNA and the cofactors described above, purified int gene product (Int) requires the participation of protein(s) encoded by the bacterial host in order to carry out integrative recombination. In the absence of this host component and spermidine, highly purified Int binds to DNA, forming specific stable complexes with DNA containing attP. Thus, Int is a specificity element for integrative recombination, recognizing at least one of the two recombining sites. We now report that, in the absence of the other components of the recombinati...

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

“…One model, but not necessarily the only model, consistent with this constraint is one in which the reaction proceeds through close association of the four strands of the two core regions, followed by strand exchange catalyzed by a topoisomerase activity of FLP protein. Such a mechanism has been previously proposed for int-catalyzed phage lambda integration (10,14) and for cre-catalyzed recombination between loxP sites of phage P1 (8). This mechanism is also consistent with the behavior of FLP protein in vitro (la, 6b, 19).…”

Isolation and characterization of intermediates in site-specific recombination.

“…In the Cre lox system (8) and in the bacteriophage X integrase system (9, 10), there is genetic evidence that site-specific recombination stimulates gene conversion of nearby markers. In both these systems, studies of linking number changes in DNA during recombination (11, 12) and the ability of the recombinases to act as type I topoisomerases (13,14) are consistent with a model of sequential single-stranded breaks and exchanges. In addition, artificial Holliday intermediates have been formed using the DNA ofthe bacteriophage X att site, and it has been demonstrated that such structures are acted upon by the recombinase Int (15).…”

Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

“…Type I topoisomerase, first described in prokaryotes by Wang (1971) and in eukaryotes by Champoux and Dulbecco (1972) and later characterized by Keller (1975a, b), change the linking number in steps of one. Although their biological function is not completely defined , this class of topoisomerase has been implicated in transcriptional (Akrigg and Cook 1980;Dynan and Burgess 1981;Sternglanz et al 1981;Weisbrod 1982;Fleischmann et al 1984 ;Gilmour et al 1986;Shastry 1986) as well as in recombinational and replicational (Kikuchi and Nash 1979 ;Sternglanz et al 1981 ; also see Gellert 1981 ;Bullock et al 1985 ;Zeng et al 1985) events. J n higher eukaryotes topoisomerase I is enriched in the nucleolus (Fleischmann et al 1984;Muller et al 1985) and may play a role in transcription of supercoiled rDNA in vitro (Garg et al 1987).…”