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Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage.

Ahmed W, Bhat AG, Leelaram MN, Menon S, Nagaraja V - Nucleic Acids Res. (2013)

Bottom Line: Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme.We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step.The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.

ABSTRACT
Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn(2+) finger motifs in the CTD. The Zn(2+) finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.

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MstopoI binds two DNA molecules. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with proteins for 10 min on ice to form complex I. Further, 60-mer unlabeled oligonucleotide was incubated with the complex I for 15 min on ice to form complex II (as depicted in the schematic). Complexes were resolved on 6% native PAGE. (A) Complex formed by the WT MstopoI, NTD and CTD (as indicated in each panel); (B) EMSA with the WT and deletant enzymes. C: 32-mer with no enzyme; complex I: protein-32 mer complex; complex II: 32 mer-protein-60 mer complex.
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gkt506-F5: MstopoI binds two DNA molecules. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with proteins for 10 min on ice to form complex I. Further, 60-mer unlabeled oligonucleotide was incubated with the complex I for 15 min on ice to form complex II (as depicted in the schematic). Complexes were resolved on 6% native PAGE. (A) Complex formed by the WT MstopoI, NTD and CTD (as indicated in each panel); (B) EMSA with the WT and deletant enzymes. C: 32-mer with no enzyme; complex I: protein-32 mer complex; complex II: 32 mer-protein-60 mer complex.

Mentions: According to sign inversion model of DNA relaxation proposed for the mechanism of action of type IA topoisomerase, binding of two DNA molecules by the enzyme—scissile and the other transfer strand, is predicted (29). Indeed, Li et al. (31) suggested the existence of at least two independent DNA-binding sites in the type IA topoisomerase, which has not been experimentally verified yet. To evaluate MstopoI binding to two DNA, mobility shift assay was carried out with oligonucleotides of different sizes. First, 5′-end-labeled, 32-mer STS oligonucleotide incubated with MstopoI formed complex I (Figure 5). Incubation of complex I with increasing concentrations of unlabeled 60-mer non-STS DNA formed complex II (supershifted band in Figure 5), demonstrating that MstopoI can simultaneously bind two different oligonucleotides. Either NTD or CTD alone formed one complex with the DNA, indicating that each component contains one DNA-binding site. The complex formed with CTD could be competed out with 60-mer non-STS oligonucleotide, whereas the NTD–DNA complex was resistant to decay, confirming that the binding mediated by the CTD was non-specific, and the NTD specifically binds STS DNA, as previously demonstrated (17). Experiments with the deletants demonstrated that the deletion of basic amino acid stretches affected the formation of complex II to varying degrees. Deletant ΔB1 was marginally affected, whereas the deletants ΔB3 and ΔB23 failed to form complex II. Notably, as expected, results are in agreement with the DNA-binding experiments (STS and non-STS DNA-binding assay, Figures 3 and 4), confirming that the MstopoI can hold two DNA simultaneously by two distinct parts of the enzyme, viz. NTD and CTD. Further, CTD binds DNA through the basic amino acid stretches in a sequence-independent manner to mediate strand passage.Figure 5.


Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage.

Ahmed W, Bhat AG, Leelaram MN, Menon S, Nagaraja V - Nucleic Acids Res. (2013)

MstopoI binds two DNA molecules. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with proteins for 10 min on ice to form complex I. Further, 60-mer unlabeled oligonucleotide was incubated with the complex I for 15 min on ice to form complex II (as depicted in the schematic). Complexes were resolved on 6% native PAGE. (A) Complex formed by the WT MstopoI, NTD and CTD (as indicated in each panel); (B) EMSA with the WT and deletant enzymes. C: 32-mer with no enzyme; complex I: protein-32 mer complex; complex II: 32 mer-protein-60 mer complex.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753633&req=5

gkt506-F5: MstopoI binds two DNA molecules. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with proteins for 10 min on ice to form complex I. Further, 60-mer unlabeled oligonucleotide was incubated with the complex I for 15 min on ice to form complex II (as depicted in the schematic). Complexes were resolved on 6% native PAGE. (A) Complex formed by the WT MstopoI, NTD and CTD (as indicated in each panel); (B) EMSA with the WT and deletant enzymes. C: 32-mer with no enzyme; complex I: protein-32 mer complex; complex II: 32 mer-protein-60 mer complex.
Mentions: According to sign inversion model of DNA relaxation proposed for the mechanism of action of type IA topoisomerase, binding of two DNA molecules by the enzyme—scissile and the other transfer strand, is predicted (29). Indeed, Li et al. (31) suggested the existence of at least two independent DNA-binding sites in the type IA topoisomerase, which has not been experimentally verified yet. To evaluate MstopoI binding to two DNA, mobility shift assay was carried out with oligonucleotides of different sizes. First, 5′-end-labeled, 32-mer STS oligonucleotide incubated with MstopoI formed complex I (Figure 5). Incubation of complex I with increasing concentrations of unlabeled 60-mer non-STS DNA formed complex II (supershifted band in Figure 5), demonstrating that MstopoI can simultaneously bind two different oligonucleotides. Either NTD or CTD alone formed one complex with the DNA, indicating that each component contains one DNA-binding site. The complex formed with CTD could be competed out with 60-mer non-STS oligonucleotide, whereas the NTD–DNA complex was resistant to decay, confirming that the binding mediated by the CTD was non-specific, and the NTD specifically binds STS DNA, as previously demonstrated (17). Experiments with the deletants demonstrated that the deletion of basic amino acid stretches affected the formation of complex II to varying degrees. Deletant ΔB1 was marginally affected, whereas the deletants ΔB3 and ΔB23 failed to form complex II. Notably, as expected, results are in agreement with the DNA-binding experiments (STS and non-STS DNA-binding assay, Figures 3 and 4), confirming that the MstopoI can hold two DNA simultaneously by two distinct parts of the enzyme, viz. NTD and CTD. Further, CTD binds DNA through the basic amino acid stretches in a sequence-independent manner to mediate strand passage.Figure 5.

Bottom Line: Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme.We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step.The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.

ABSTRACT
Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn(2+) finger motifs in the CTD. The Zn(2+) finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.

Show MeSH
Related in: MedlinePlus