Properties of a mutant Cre protein that alters the topological linkage of recombination products.
The bacteriophage P1 Cre-loxP site-specific recombination system consists of two components: the Cre recombinase protein and the loxP DNA sequence where recombination takes place. We report here on the analysis of a mutation in the cre structural gene that produces a mutant protein with altered recombination properties. The mutant protein, Cre111, carries out recombination at a much slower rate than the wild-type Cre protein. To determine why the reaction is slow, we have examined a number of activities associated with Cre-mediated recombination. Our results indicate that the binding of Cre111 to the loxP site is comparable to wild-type Cre. Furthermore, the rate at which Cre111 resolves Holliday structures, an intermediate in this recombination reaction, is also comparable to wild-type Cre. Thus, DNA binding and resolution of the intermediate are not affected, suggesting that either synapsis, the process of bringing two lox sites together, or the first strand exchange event, could be affected in the mutant protein. The types of DNA products formed following recombination of a supercoiled substrate can reflect mechanisms of synapsis. Wild-type Cre generates mainly topologically unlinked and unknotted circular products. This suggests that the wild-type protein brings two lox sites together in a way that excludes the entanglement of supercoils present in the substrate DNA. In contrast, when Cre111 recombines a supercoiled molecule it generates many complicated catenanes and knotted DNA products. Presumably, the supercoils present in the DNA substrate are being trapped in the reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)
Abremski
K
K
E.I. du Pont de Nemours & C Co., Inc., Central Research and Development Department, Wilmington, Delaware 19898.
Frommer
B
B
Wierzbicki
A
A
Hoess
R H
RH
D003090:Coliphages / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D019426:Integrases; D007700:Kinetics; D009154:Mutation; D011995:Recombination, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases