sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recent I y we developed a positive selection vector for analysis of plasmid recombination events in B.subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Severallines of evidence suggest that singlestrand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resembie potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase lof B.subtilis: S'-A/TCATA/T TAA/~/TA-3'. and expression vectors for bacilli to exploit their full potenrial is the frequently observed genetic instability , in particular of foreign DNA. lllegitimate recombination, which is a major source of genome rearrangements in nature (for reviews see 2,3), was shown to underlie structural plasrnid instability in numerous cases. These rccA-independent events can occur in several different ways. Recombinarion between short directly repeated sequences (DRs) of 3-20 b p can result fiom either copy choice recombination or DNA breakage and reunion mechanisffis (for reviews see 3,4). Breakage and reunion events also underlie recombination between sequences that share no homology; these processes are thought to involve certain DNA handling enzymes, such as topoisomerases, origin nicking enzymes, transposases, invertases and site-specific recombinases. DNA topoisomerases are pivotal enzymes in the control of topological transitions of DNA. A role of these enzymes in illegitirnate recombination has been demonstrated by several authors (5-11). These events involve uncoupling of the nicking and closing reactions catalysed by DNA topoisomerases and result in joining of umelated ends, producing recombinant molecules. In addition, specialized type I topoisomerases, like phage A integrase (12) and PI Cre recombinase (13), mediate DNA strand transfer in site-specific recombination reactions. Recently Z hu and Schiestl ( 14) demonstrated that overproduction of topoisoffierase I (Topo I) in Saccharomyccs ccrcvisiac increased the levelof illegitimate integration in transformation studies with non-homologous DNA.