Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition 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. Recently 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. Several lines of evidence suggest that single-strand 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 resemble 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 I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.
Meima
R
R
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Haan
G J
GJ
Venema
G
G
Bron
S
S
de Jong
S
S
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000032:analysis; D018628:Gene Dosage; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D017384:Sequence Deletion; D013379:Substrate Specificity
D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I