Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site.
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.
Leo
Elisabetta
E
Molecular Genetics Group, Department of Basic Medical Sciences-Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom.
Gould
Katherine A
KA
Pan
Xiao-Su
XS
Capranico
Giovanni
G
Sanderson
Mark R
MR
Palumbo
Manlio
M
Fisher
L Mark
LM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D027101:DNA Topoisomerase IV / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D024841:Fluoroquinolones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase