Limits...
Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.

Pan XS, Dias M, Palumbo M, Fisher LM - Nucleic Acids Res. (2008)

Bottom Line: CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci.Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones.The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

Show MeSH

Related in: MedlinePlus

CL induces DNA modification and irreversible/reversible DNA breakage at gyrase cleavage sites. (A) Probing CL modification and cleavage reversibility. The S fragment, 33P-labelled at the 5′ end of the TOP or BOTTOM strand, was incubated with S. pneumoniae gyrase and 1 mM ATP (lanes 2–12) in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (lanes 8–12). Samples in lanes 5 and 10, 6 and 11 and 7 and 12 were further incubated with 0.6 M NaCl for 15 min (denoted by S), with 35 mM EDTA for 15 min (E), or heating to 65°C for 15 min (T), respectively. After cleavage induction with SDS and proteinase K, DNA was precipitated with ethanol. Cleavage products in lanes 4–7 and 9–12 were further incubated with 1-M piperidine at 90°C for 30 min and then lyophilized. Reaction products were loaded and run on 6% denaturing polyacrylamide gels alongside the G + A Maxam–Gilbert chemical sequencing products, and the ACGT dideoxy sequencing products obtained using the same 5′ end-labelled oligonucleotide primers. Lane 1 is untreated substrate DNA. Lane 2 is a control for gyrase-processed DNA. Lanes 3 and 8 are cleavage products that were not treated with piperidine. DNA cleavage products that undergo shortening after hot piperidine are denoted by filled circles; those unaffected by piperidine are labelled with open circles. Gemifloxacin cleavage products were uniformly unaffected by hot piperidine (compare lanes 8 and 9) and were formed reversibly (lanes 10–12). Several new bands appearing after EDTA treatment (TOP strand, lane 6) may arise from sealing of sites proximal to the 5′ end revealing distal cleavage sites. (B) Mapping CL-gyrase cleavage sites in the S fragment. Arrowheads and arrows show cleavage sites stimulated by CL and gemifloxacin, respectively. Filled and open arrowheads denote sites of irreversible and reversible cleavage, respectively. Filled and open circles denote CL sites at which the 3′ nucleotide was released by hot piperidine or was wholly (or partially) unaffected by piperidine treatment, respectively. The large arrowheads and circles denote the most efficient cleavage site. Different to the 4-bp gyrase stagger, two sites with an apparent 2-bp overhang likely arise from closely spaced single cuts. Numbers refer to nucleotide positions in the S. pneumoniae parE gene (31) counting from the first nucleotide of the initiation codon ATG. Horizontal arrows denote the sequences of forward (S6398) and reverse oligonucleotides (S6399) used in PCR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553588&req=5

Figure 4: CL induces DNA modification and irreversible/reversible DNA breakage at gyrase cleavage sites. (A) Probing CL modification and cleavage reversibility. The S fragment, 33P-labelled at the 5′ end of the TOP or BOTTOM strand, was incubated with S. pneumoniae gyrase and 1 mM ATP (lanes 2–12) in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (lanes 8–12). Samples in lanes 5 and 10, 6 and 11 and 7 and 12 were further incubated with 0.6 M NaCl for 15 min (denoted by S), with 35 mM EDTA for 15 min (E), or heating to 65°C for 15 min (T), respectively. After cleavage induction with SDS and proteinase K, DNA was precipitated with ethanol. Cleavage products in lanes 4–7 and 9–12 were further incubated with 1-M piperidine at 90°C for 30 min and then lyophilized. Reaction products were loaded and run on 6% denaturing polyacrylamide gels alongside the G + A Maxam–Gilbert chemical sequencing products, and the ACGT dideoxy sequencing products obtained using the same 5′ end-labelled oligonucleotide primers. Lane 1 is untreated substrate DNA. Lane 2 is a control for gyrase-processed DNA. Lanes 3 and 8 are cleavage products that were not treated with piperidine. DNA cleavage products that undergo shortening after hot piperidine are denoted by filled circles; those unaffected by piperidine are labelled with open circles. Gemifloxacin cleavage products were uniformly unaffected by hot piperidine (compare lanes 8 and 9) and were formed reversibly (lanes 10–12). Several new bands appearing after EDTA treatment (TOP strand, lane 6) may arise from sealing of sites proximal to the 5′ end revealing distal cleavage sites. (B) Mapping CL-gyrase cleavage sites in the S fragment. Arrowheads and arrows show cleavage sites stimulated by CL and gemifloxacin, respectively. Filled and open arrowheads denote sites of irreversible and reversible cleavage, respectively. Filled and open circles denote CL sites at which the 3′ nucleotide was released by hot piperidine or was wholly (or partially) unaffected by piperidine treatment, respectively. The large arrowheads and circles denote the most efficient cleavage site. Different to the 4-bp gyrase stagger, two sites with an apparent 2-bp overhang likely arise from closely spaced single cuts. Numbers refer to nucleotide positions in the S. pneumoniae parE gene (31) counting from the first nucleotide of the initiation codon ATG. Horizontal arrows denote the sequences of forward (S6398) and reverse oligonucleotides (S6399) used in PCR.

Mentions: In the absence of topoisomerases, it is known that the epoxide moiety of CL reacts at N-7 of unpaired guanines (6). Incubation over extended times (24–48 h) leads to loss of the N-7 alkylated base and strand scission at the abasic site, a process used in the Maxam–Gilbert DNA sequencing protocol and facilitated by hot piperidine (32). To determine whether CL modifies bases exposed in the gyrase cleavage complex, we compared the mobility of cleavage products from the TOP and BOTTOM strands of the S fragment (Figure 4A) before (lanes 3) and after hot piperidine (lanes 4). A number of fragments (denoted by filled circles on Figure 4A) suffered a 1-nt shift in mobility after hot piperidine consistent with the loss of a modified nucleotide at their extreme 3′ end, i.e. the nucleotide at −1. After hot piperidine, these fragments co-migrated with the Maxam–Gilbert ladder which have 3′ phosphate termini, Shifted fragments invariably arose from scission at −1G and their production could not be reversed with salt, EDTA or high temperature (Figure 4A, lanes 5–7). However, the mobility of other cleavage fragments (denoted by open circles in Figure 4A) was not affected by hot piperidine (lanes 3 and 4). These fragments arose from cleavage at sites with A, C, T or even G at −1, and invariably their production could be at least partially reversed with EDTA (lanes 6), though not with salt or heat (lanes 5 and 7). Control experiments showed that gemifloxacin-promoted cleavage products were universally unaffected by piperidine consistent with the known non-covalent interaction of quinolones with the cleavage complex (lanes 8 and 9). Moreover, cleavage by gemifloxacin (lane 8), a high-affinity quinolone (26), was partially reversed with high salt (lane 10) and fully reversed with EDTA and heat (lanes 11 and 12), consistent with sealing of unmodified 3′ OH breaks. Thus, in contrast to quinolones, it appears that CL stimulates both irreversible and reversible gyrase cleavage of the S fragment with evidence of covalent modification at some guanine sites.Figure 4.


Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.

Pan XS, Dias M, Palumbo M, Fisher LM - Nucleic Acids Res. (2008)

CL induces DNA modification and irreversible/reversible DNA breakage at gyrase cleavage sites. (A) Probing CL modification and cleavage reversibility. The S fragment, 33P-labelled at the 5′ end of the TOP or BOTTOM strand, was incubated with S. pneumoniae gyrase and 1 mM ATP (lanes 2–12) in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (lanes 8–12). Samples in lanes 5 and 10, 6 and 11 and 7 and 12 were further incubated with 0.6 M NaCl for 15 min (denoted by S), with 35 mM EDTA for 15 min (E), or heating to 65°C for 15 min (T), respectively. After cleavage induction with SDS and proteinase K, DNA was precipitated with ethanol. Cleavage products in lanes 4–7 and 9–12 were further incubated with 1-M piperidine at 90°C for 30 min and then lyophilized. Reaction products were loaded and run on 6% denaturing polyacrylamide gels alongside the G + A Maxam–Gilbert chemical sequencing products, and the ACGT dideoxy sequencing products obtained using the same 5′ end-labelled oligonucleotide primers. Lane 1 is untreated substrate DNA. Lane 2 is a control for gyrase-processed DNA. Lanes 3 and 8 are cleavage products that were not treated with piperidine. DNA cleavage products that undergo shortening after hot piperidine are denoted by filled circles; those unaffected by piperidine are labelled with open circles. Gemifloxacin cleavage products were uniformly unaffected by hot piperidine (compare lanes 8 and 9) and were formed reversibly (lanes 10–12). Several new bands appearing after EDTA treatment (TOP strand, lane 6) may arise from sealing of sites proximal to the 5′ end revealing distal cleavage sites. (B) Mapping CL-gyrase cleavage sites in the S fragment. Arrowheads and arrows show cleavage sites stimulated by CL and gemifloxacin, respectively. Filled and open arrowheads denote sites of irreversible and reversible cleavage, respectively. Filled and open circles denote CL sites at which the 3′ nucleotide was released by hot piperidine or was wholly (or partially) unaffected by piperidine treatment, respectively. The large arrowheads and circles denote the most efficient cleavage site. Different to the 4-bp gyrase stagger, two sites with an apparent 2-bp overhang likely arise from closely spaced single cuts. Numbers refer to nucleotide positions in the S. pneumoniae parE gene (31) counting from the first nucleotide of the initiation codon ATG. Horizontal arrows denote the sequences of forward (S6398) and reverse oligonucleotides (S6399) used in PCR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553588&req=5

Figure 4: CL induces DNA modification and irreversible/reversible DNA breakage at gyrase cleavage sites. (A) Probing CL modification and cleavage reversibility. The S fragment, 33P-labelled at the 5′ end of the TOP or BOTTOM strand, was incubated with S. pneumoniae gyrase and 1 mM ATP (lanes 2–12) in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (lanes 8–12). Samples in lanes 5 and 10, 6 and 11 and 7 and 12 were further incubated with 0.6 M NaCl for 15 min (denoted by S), with 35 mM EDTA for 15 min (E), or heating to 65°C for 15 min (T), respectively. After cleavage induction with SDS and proteinase K, DNA was precipitated with ethanol. Cleavage products in lanes 4–7 and 9–12 were further incubated with 1-M piperidine at 90°C for 30 min and then lyophilized. Reaction products were loaded and run on 6% denaturing polyacrylamide gels alongside the G + A Maxam–Gilbert chemical sequencing products, and the ACGT dideoxy sequencing products obtained using the same 5′ end-labelled oligonucleotide primers. Lane 1 is untreated substrate DNA. Lane 2 is a control for gyrase-processed DNA. Lanes 3 and 8 are cleavage products that were not treated with piperidine. DNA cleavage products that undergo shortening after hot piperidine are denoted by filled circles; those unaffected by piperidine are labelled with open circles. Gemifloxacin cleavage products were uniformly unaffected by hot piperidine (compare lanes 8 and 9) and were formed reversibly (lanes 10–12). Several new bands appearing after EDTA treatment (TOP strand, lane 6) may arise from sealing of sites proximal to the 5′ end revealing distal cleavage sites. (B) Mapping CL-gyrase cleavage sites in the S fragment. Arrowheads and arrows show cleavage sites stimulated by CL and gemifloxacin, respectively. Filled and open arrowheads denote sites of irreversible and reversible cleavage, respectively. Filled and open circles denote CL sites at which the 3′ nucleotide was released by hot piperidine or was wholly (or partially) unaffected by piperidine treatment, respectively. The large arrowheads and circles denote the most efficient cleavage site. Different to the 4-bp gyrase stagger, two sites with an apparent 2-bp overhang likely arise from closely spaced single cuts. Numbers refer to nucleotide positions in the S. pneumoniae parE gene (31) counting from the first nucleotide of the initiation codon ATG. Horizontal arrows denote the sequences of forward (S6398) and reverse oligonucleotides (S6399) used in PCR.
Mentions: In the absence of topoisomerases, it is known that the epoxide moiety of CL reacts at N-7 of unpaired guanines (6). Incubation over extended times (24–48 h) leads to loss of the N-7 alkylated base and strand scission at the abasic site, a process used in the Maxam–Gilbert DNA sequencing protocol and facilitated by hot piperidine (32). To determine whether CL modifies bases exposed in the gyrase cleavage complex, we compared the mobility of cleavage products from the TOP and BOTTOM strands of the S fragment (Figure 4A) before (lanes 3) and after hot piperidine (lanes 4). A number of fragments (denoted by filled circles on Figure 4A) suffered a 1-nt shift in mobility after hot piperidine consistent with the loss of a modified nucleotide at their extreme 3′ end, i.e. the nucleotide at −1. After hot piperidine, these fragments co-migrated with the Maxam–Gilbert ladder which have 3′ phosphate termini, Shifted fragments invariably arose from scission at −1G and their production could not be reversed with salt, EDTA or high temperature (Figure 4A, lanes 5–7). However, the mobility of other cleavage fragments (denoted by open circles in Figure 4A) was not affected by hot piperidine (lanes 3 and 4). These fragments arose from cleavage at sites with A, C, T or even G at −1, and invariably their production could be at least partially reversed with EDTA (lanes 6), though not with salt or heat (lanes 5 and 7). Control experiments showed that gemifloxacin-promoted cleavage products were universally unaffected by piperidine consistent with the known non-covalent interaction of quinolones with the cleavage complex (lanes 8 and 9). Moreover, cleavage by gemifloxacin (lane 8), a high-affinity quinolone (26), was partially reversed with high salt (lane 10) and fully reversed with EDTA and heat (lanes 11 and 12), consistent with sealing of unmodified 3′ OH breaks. Thus, in contrast to quinolones, it appears that CL stimulates both irreversible and reversible gyrase cleavage of the S fragment with evidence of covalent modification at some guanine sites.Figure 4.

Bottom Line: CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci.Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones.The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

Show MeSH
Related in: MedlinePlus