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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.

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Substitution of −1 guanines at the pBR322 ‘1073 site’ with adenine, cytosine or thymine abolishes gyrase DNA cleavage stimulated by CL but not by gemifloxacin. The 295-bp pBR322 fragment 5′ 33P-labelled on the bottom strand was amplified by PCR from pBR322 plasmids carrying the wild-type site (G at both −1 positions, indicated by WT) or mutant sites bearing A, C or T at both −1 positions (denoted by −1A, −1C and −1T). The fragments were used in a DNA cleavage assay with gyrase and 1 mM ATP and either 100 μM gemifloxacin (lanes 1) or 200 μM CL (lanes 2 and 3). DNA was recovered by ethanol precipitation and run on an 6% denaturing polyacrylamide gel alongside ACGT chain termination DNA sequencing products obtained from a wild-type DNA template and G+A Maxam–Gilbert chemical sequencing products generated for each of the four substrates. Prior to electrophoresis, one set of CL cleavage products was treated with hot piperidine (lanes 3). Open circle denotes cleavage product derived from the major cleavage site.
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Figure 6: Substitution of −1 guanines at the pBR322 ‘1073 site’ with adenine, cytosine or thymine abolishes gyrase DNA cleavage stimulated by CL but not by gemifloxacin. The 295-bp pBR322 fragment 5′ 33P-labelled on the bottom strand was amplified by PCR from pBR322 plasmids carrying the wild-type site (G at both −1 positions, indicated by WT) or mutant sites bearing A, C or T at both −1 positions (denoted by −1A, −1C and −1T). The fragments were used in a DNA cleavage assay with gyrase and 1 mM ATP and either 100 μM gemifloxacin (lanes 1) or 200 μM CL (lanes 2 and 3). DNA was recovered by ethanol precipitation and run on an 6% denaturing polyacrylamide gel alongside ACGT chain termination DNA sequencing products obtained from a wild-type DNA template and G+A Maxam–Gilbert chemical sequencing products generated for each of the four substrates. Prior to electrophoresis, one set of CL cleavage products was treated with hot piperidine (lanes 3). Open circle denotes cleavage product derived from the major cleavage site.

Mentions: The role of the −1 nt in CL action was further investigated using directed mutagenesis of the major pBR322 gyrase site at 1073, a site bearing G at both −1 positions (Figure 5B) and which was cleaved some 10× more efficiently than other sites, e.g. in the S fragment. Given that sites exhibiting a single −1G can be cleaved (Table 1, Figures 4B and 5B), we constructed mutants of the pBR322 site in which both −1 positions (G1073 and complementary strand G1078) were altered to either A, C or T nucleotides. The sites, contained in end-labelled 295-bp PCR products, were used as cleavage substrates for gyrase in the presence of gemifloxacin or CL, and the products were displayed on a denaturing polyacrylamide gel (Figure 6). Gemifloxacin promoted efficient double-stranded DNA cleavage at the wild-type (WT) site and its −1A, −1C and −1T mutants (lanes 1) producing the expected fragment (circles in Figure 6), thereby showing that the mutant sites are still recognized by gyrase. CL induced cleavage at the WT site to produce a DNA fragment that co-migrated with the gemifloxacin-stabilized fragment and with the appropriate dideoxy DNA sequencing product (WT, lanes 1 and 2). However, this fragment was absent for CL cleavage of the mutant −1A, −1C and −1T substrates (lanes 2). Instead, enhanced cleavage was seen at nearby sites i.e. 3′ of G1079 (smaller fragment) and G1075 (larger fragment) (Figure 6, −1A, −1C and −1T, lanes 2) and both fragments exhibited a one-base shortening on piperidine treatment (lanes 3). These mutagenesis data provide the first direct evidence that CL-induced gyrase cleavage of DNA is prevented when G is absent from both −1 positions.Figure 6.


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)

Substitution of −1 guanines at the pBR322 ‘1073 site’ with adenine, cytosine or thymine abolishes gyrase DNA cleavage stimulated by CL but not by gemifloxacin. The 295-bp pBR322 fragment 5′ 33P-labelled on the bottom strand was amplified by PCR from pBR322 plasmids carrying the wild-type site (G at both −1 positions, indicated by WT) or mutant sites bearing A, C or T at both −1 positions (denoted by −1A, −1C and −1T). The fragments were used in a DNA cleavage assay with gyrase and 1 mM ATP and either 100 μM gemifloxacin (lanes 1) or 200 μM CL (lanes 2 and 3). DNA was recovered by ethanol precipitation and run on an 6% denaturing polyacrylamide gel alongside ACGT chain termination DNA sequencing products obtained from a wild-type DNA template and G+A Maxam–Gilbert chemical sequencing products generated for each of the four substrates. Prior to electrophoresis, one set of CL cleavage products was treated with hot piperidine (lanes 3). Open circle denotes cleavage product derived from the major cleavage site.
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Figure 6: Substitution of −1 guanines at the pBR322 ‘1073 site’ with adenine, cytosine or thymine abolishes gyrase DNA cleavage stimulated by CL but not by gemifloxacin. The 295-bp pBR322 fragment 5′ 33P-labelled on the bottom strand was amplified by PCR from pBR322 plasmids carrying the wild-type site (G at both −1 positions, indicated by WT) or mutant sites bearing A, C or T at both −1 positions (denoted by −1A, −1C and −1T). The fragments were used in a DNA cleavage assay with gyrase and 1 mM ATP and either 100 μM gemifloxacin (lanes 1) or 200 μM CL (lanes 2 and 3). DNA was recovered by ethanol precipitation and run on an 6% denaturing polyacrylamide gel alongside ACGT chain termination DNA sequencing products obtained from a wild-type DNA template and G+A Maxam–Gilbert chemical sequencing products generated for each of the four substrates. Prior to electrophoresis, one set of CL cleavage products was treated with hot piperidine (lanes 3). Open circle denotes cleavage product derived from the major cleavage site.
Mentions: The role of the −1 nt in CL action was further investigated using directed mutagenesis of the major pBR322 gyrase site at 1073, a site bearing G at both −1 positions (Figure 5B) and which was cleaved some 10× more efficiently than other sites, e.g. in the S fragment. Given that sites exhibiting a single −1G can be cleaved (Table 1, Figures 4B and 5B), we constructed mutants of the pBR322 site in which both −1 positions (G1073 and complementary strand G1078) were altered to either A, C or T nucleotides. The sites, contained in end-labelled 295-bp PCR products, were used as cleavage substrates for gyrase in the presence of gemifloxacin or CL, and the products were displayed on a denaturing polyacrylamide gel (Figure 6). Gemifloxacin promoted efficient double-stranded DNA cleavage at the wild-type (WT) site and its −1A, −1C and −1T mutants (lanes 1) producing the expected fragment (circles in Figure 6), thereby showing that the mutant sites are still recognized by gyrase. CL induced cleavage at the WT site to produce a DNA fragment that co-migrated with the gemifloxacin-stabilized fragment and with the appropriate dideoxy DNA sequencing product (WT, lanes 1 and 2). However, this fragment was absent for CL cleavage of the mutant −1A, −1C and −1T substrates (lanes 2). Instead, enhanced cleavage was seen at nearby sites i.e. 3′ of G1079 (smaller fragment) and G1075 (larger fragment) (Figure 6, −1A, −1C and −1T, lanes 2) and both fragments exhibited a one-base shortening on piperidine treatment (lanes 3). These mutagenesis data provide the first direct evidence that CL-induced gyrase cleavage of DNA is prevented when G is absent from both −1 positions.Figure 6.

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