Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific.

Abstract

Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.

• Authors: S Shuman
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 88 Issue 22 Pg. 10104-8 (Nov 15 1991) ISSN: 0027-8424 [Print] United States
• PMID: 1658796 (Publication Type: Journal Article)

Chemical References

• DNA, Viral
• Recombinant Proteins
• DNA Topoisomerases, Type I

Topics

• Bacteriophage lambda (genetics)
• Base Sequence
• Cloning, Molecular
• DNA Topoisomerases, Type I (genetics, metabolism)
• DNA, Viral (genetics)
• Escherichia coli (genetics)
• Lysogeny
• Models, Genetic
• Molecular Sequence Data
• Molecular Weight
• Recombinant Proteins (metabolism)
• Recombination, Genetic
• Vaccinia virus (enzymology, genetics)