Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.

Abstract

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and -galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency ofTn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in SalmoneUa typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.Escherichia coli DNA topoisomerase I, also known as w protein, has been the subject of extensive studies (for a review, see ref.1). The enzyme is a single polypeptide ofabout 110,000 daltons. In vitro it catalyzes the relaxation of negatively supercoiled DNA (2), the knotting and unknotting of single-stranded DNA rings (3), the linking of single-stranded DNA rings of complementary sequences into intertwined duplex rings (4), and the catenation and decatenation of duplex DNA rings when at least one member of a pair of participating duplex rings has a singlechain scission (5). The catalysis of these topological isomerization reactions by the enzyme is believed to involve transient single-stranded breakage of DNA phosphodiester bonds (1).In contrast to the extensive in vitro studies, there has been little work on the functions of the enzyme in vivo. We have therefore undertaken the isolation and characterization of mutants deficient in DNA topoisomerase I activity. In this communication, we report the identification of a number of mutations in the structural gene for the enzyme and the genetic mapping ofthis gene, which we have termed top, on the E. coli chromosome. Roles of the enzyme in the regulation of transcription ofa number ofoperons and in the transposition of several transposons are also implicated based on our initial characterization of the mutants.MATERIALS AND METHODS Bacterial Strains. All strains used were E. coli K-12 derivatives. The strain used to produce the collection oftemperaturesensitive mutants was PA3092 F-thr leu thi argH thyA his trp lacYl mtl xyl malA mel tonA str supE. Two mutants from this collection were found to have top mutations, JE10010 topl0 and JE10250 top250 (6). Other strains used include: P4X8 Hfr met; KV385 met lacYl xyl mtl str topl0; PLK831 F-trpE pyrF gal-25 nirA strA195; JTT1, a trp+ top+...

Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

“…Type I topoisomerase, first described in prokaryotes by Wang (1971) and in eukaryotes by Champoux and Dulbecco (1972) and later characterized by Keller (1975a, b), change the linking number in steps of one. Although their biological function is not completely defined , this class of topoisomerase has been implicated in transcriptional (Akrigg and Cook 1980;Dynan and Burgess 1981;Sternglanz et al 1981;Weisbrod 1982;Fleischmann et al 1984 ;Gilmour et al 1986;Shastry 1986) as well as in recombinational and replicational (Kikuchi and Nash 1979 ;Sternglanz et al 1981 ; also see Gellert 1981 ;Bullock et al 1985 ;Zeng et al 1985) events. J n higher eukaryotes topoisomerase I is enriched in the nucleolus (Fleischmann et al 1984;Muller et al 1985) and may play a role in transcription of supercoiled rDNA in vitro (Garg et al 1987).…”

Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling.

“…In contrast, topoisomerase I is not essential: deletion mutants are viable (22). However, topoisomerase I deletion mutants of Escherichia coli grow well only because they have acquired compensatory mutations (7,23).…”

Viability of Escherichia coli topA Mutants Lacking DNA Topoisomerase I

“…Shortly after the identification of a set of viable E. coli ⌬topA mutants (25), it was found that the ⌬topA locus of these mutants could not be readily transduced into strain PLK831 (⌬trpE63 pyrF287 nirA trpR72 iclR7 gal25 rpsL195) by phage P1 (26,27). The same recipient strain became more easily transduced, however, if it had acquired a compensatory change within certain regions of the E. coli chromosome, including gyrA and gyrB, which encode the two subunits of gyrase, and a region containing tolC, which encodes an outer membrane transporter (26 -29).…”