Abstract
We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.
Highlights
From the Departmentof Molecular Biology, The Instituteof Medical Science, University of Tokyo, P.0
We have shown that there is either no homology orthere are 1-5-base pair homologies between the parental DNAs in seven combinations of X recombination sites, indicating that homology is not essential for the recombination.we have shown an association of the recombination sites withthe topoisomerase cleavage sites,indicating that a capacity of the topoisomeraseto make a transient double-strandedbreak in DNA plays a role inthe illegitimate recombination
One type of rearrangement is produced by illegitimate recombination events.These recombinations are distinguishable from homologous recombination which requires extensive homology or from site-specific recombination which requires specific sites
Summary
BacteriaB,acteriophagea,nPdlasmiSdtrains-The bacterial strains used were all derivatives of E. coli K12. X imm434cI Sam Ram int red were used as parental phage for recombination (16). They arecalled Xb538DF and XSR, respectively, in this paper. M13mp was used for cloning of DNA fragments of phage X and for. PUC9wasused for cloning of recombination junctions of X DNA. The sequence of the recombination junction was deter- DNAs. mined by the dideoxy method. The following restriction fragments containing recombination junctions were cloned into pUC9 and used for sequencing: HpaI fragment of5600 bp from XMM3; HindIII-ScaI fragment of 2000 bp from XMM.I; ScaI-SmaI fragment of3400 bp from XMM7; BamHI-SmaI fragment of3600 bp from XMM8; MluI-PuuII fragment of 1000bp from XMM14; PstI fragment of 2600 bp from XMM18. DNA fragments containing recombination sites were recovered
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