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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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Related in: MedlinePlus

DNA-binding ability of the MstopoI deletants. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with varying concentrations of enzymes, as mentioned in each panel. DNA–protein complexes were quantitated, and DNA-binding affinity of WT and its deletants are represented in the form of their respective Kd value.
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gkt506-F3: DNA-binding ability of the MstopoI deletants. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with varying concentrations of enzymes, as mentioned in each panel. DNA–protein complexes were quantitated, and DNA-binding affinity of WT and its deletants are represented in the form of their respective Kd value.

Mentions: In an earlier study, we showed that the CTD binds to DNA in a non-specific manner (17). From the results described earlier in the text, it appeared that basic amino acids could confer DNA-binding property to the CTD. To test this, EMSA was carried out using 32-mer single-stranded STS oligonucleotide and various CTD deletant enzymes described earlier in the text. From the representative Kd values, it is apparent that the deletion of basic amino acid stretches reduced the DNA-binding affinity of the enzyme (Figure 3). Despite weaker binding of the mutants, their cleavage and religation activities were comparable with the WT enzyme (Supplementary Figures S3 and S4). From these data, one can surmise that the stretches in the CTD do not influence the binding of MstopoI to the scissile DNA. Instead, their role could be in holding the non-scissile (non-STS) complementary DNA required for strand passage. To assess their role in non-specific DNA capture, DNA-binding ability of deletants was determined with the non-STS DNA. As the binding of NTD to non-STS DNA is extremely low (17), the complex formed by the MstopoI with this DNA would mainly be by the contribution from the CTD. From the EMSA results (Figure 4A), it is evident that the ΔB1and ΔB3 were affected in non-specific DNA binding significantly, and the binding of ΔB13 and ΔB23 to DNA was completely abolished.Figure 3.


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)

DNA-binding ability of the MstopoI deletants. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with varying concentrations of enzymes, as mentioned in each panel. DNA–protein complexes were quantitated, and DNA-binding affinity of WT and its deletants are represented in the form of their respective Kd value.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt506-F3: DNA-binding ability of the MstopoI deletants. EMSAs were carried out by incubating 5′-end-labeled 32-mer oligonucleotide containing STS with varying concentrations of enzymes, as mentioned in each panel. DNA–protein complexes were quantitated, and DNA-binding affinity of WT and its deletants are represented in the form of their respective Kd value.
Mentions: In an earlier study, we showed that the CTD binds to DNA in a non-specific manner (17). From the results described earlier in the text, it appeared that basic amino acids could confer DNA-binding property to the CTD. To test this, EMSA was carried out using 32-mer single-stranded STS oligonucleotide and various CTD deletant enzymes described earlier in the text. From the representative Kd values, it is apparent that the deletion of basic amino acid stretches reduced the DNA-binding affinity of the enzyme (Figure 3). Despite weaker binding of the mutants, their cleavage and religation activities were comparable with the WT enzyme (Supplementary Figures S3 and S4). From these data, one can surmise that the stretches in the CTD do not influence the binding of MstopoI to the scissile DNA. Instead, their role could be in holding the non-scissile (non-STS) complementary DNA required for strand passage. To assess their role in non-specific DNA capture, DNA-binding ability of deletants was determined with the non-STS DNA. As the binding of NTD to non-STS DNA is extremely low (17), the complex formed by the MstopoI with this DNA would mainly be by the contribution from the CTD. From the EMSA results (Figure 4A), it is evident that the ΔB1and ΔB3 were affected in non-specific DNA binding significantly, and the binding of ΔB13 and ΔB23 to DNA was completely abolished.Figure 3.

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