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K Abremski

Publications and source records attributed to K Abremski.

28 records · Page 2Linked to original sources

The nature of the interaction of the P1 recombinase Cre with the recombining site loxP.

The experiments reported here begin to define the molecular nature of the Cre-loxP interaction. It is instructive to compare some of the features of this system with the two other well-characterized site-specific recombination systems of phage lambda and transposon Tn3. In terms of the DNA required for a functional recombining site, the Cre-loxP system appears to be the simplest of the three, requiring only 34 bp of sequence to bind the recombinase. In contrast, both lambda and Tn3 systems have multiple binding sites for their respective recombinases (Hsu et al. 1980; Grindley et al. 1982). It is believed that these additional binding sites, although not the sites of cleavage and strand exchange, are somehow important in bringing the DNA into a suitable configuration necessary for recombination (Better et al. 1982; Grindley et al. 1982; Pollock and Nash 1983). DNA conformation, i.e., whether the DNA substrate is supercoiled or linear, also appears to play a more important role in both of these systems, whereas the Cre-loxP system works efficiently, irrespective of the DNA conformation. All of the recombinases mentioned above create staggered cuts in the DNA during the process of strand exchange and form a covalent attachment to the DNA at the site of cleavage (Reed and Grindley 1981; Craig and Nash 1983b; R.H. Hoess and K. Abremski, in prep.). It should be pointed out that covalent attachment to the DNA is a general feature that these recombinases share with topoisomerases (Gellert 1981). The nature of the cuts made in the DNA is more similar for Int and Cre than for Tn3 resolvase.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination.

Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre. A number of lambda and plasmid substrates containing two loxP sites have been constructed. Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp. In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently. The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles. The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.

Bacteriophage lambda↗

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product.

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.

Bacteriophage lambda↗

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination.

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.

Bacteriophage lambda↗