Abstract
Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.
Highlights
Anti-pneumococcal Quinolones Promote Site-specific DNA Cleavage by Topo IV and Gyrase—To investigate DNA breakage by pneumococcal topo IV and gyrase, we used four fluoroquinolones either clinically approved or in clinical use for pneumonia and other infections caused by S. pneumoniae, namely gemifloxacin, gatifloxacin, moxifloxacin, and levofloxacin (Fig. 1)
Except for minor differences in some band intensities, the four quinolones promoted gyrase-induced DNA cleavage at the same spectrum of sites. These findings were seen using other plasmid substrates and other quinolones, e.g. ciprofloxacin, clinafloxacin, and sparfloxacin. It appears that differences in quinolone structure do not affect the specificity of DNA breakage by either topo IV or gyrase
The Major pBR322 Site for E. coli Gyrase Is Efficiently Cleaved by S. pneumoniae Gyrase and Topo IV—To examine whether pneumococcal type II enzymes could act at E. coli gyrase cleavage sites, we investigated cleavage at the strong site of oxolinic acid-promoted gyrase cleavage that maps at nucleotide position 990 in pBR322 [14, 30]
Summary
Type IIA DNA topoisomerases mediate the ATP-dependent transport of one DNA helix through another DNA segment via a transient enzyme-bridged double-strand break [1,2,3,4]. Segments may belong to different molecules allowing decatenation of interlocked DNA It appears that wrapping of a 120 –150-bp DNA region encompassing the DNA breakage site on the G segment favors intramolecular DNA strand passage producing DNA supercoiling [13,14,15,16], whereas the absence of DNA wrapping on topo IV allows efficient intermolecular DNA passage and chromosome decatenation. Transient double-strand DNA breakage by topo IV and gyrase proceeds via an enzyme-DNA intermediate termed the “cleavage complex” involving a 4-bp staggered break and covalent attachment of ParC (GyrA) subunits to each 5Ј DNA end through a phosphotyrosine linkage (with Tyr-122 in E. coli GyrA) [2, 5]. It is clear that the sequence determinants for DNA breakage have yet to be convincingly established for either topo IV or gyrase
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