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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

Overexpression of the deletant enzyme results in growth defect. Exponential phase cultures of M. smegmatis mc2 155 and overexpression strains were diluted to O.D.595 nm = 0.05 in Middlebrook7H9 broth and incubated at 37°C with continuous shaking. The cultures were grown for 40 h, and the O.D.595nm was measured at every 2 h interval. The figure shows the mean O.D.595nmvalues obtained in three independent experiments. Bar represents the SD.
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gkt506-F6: Overexpression of the deletant enzyme results in growth defect. Exponential phase cultures of M. smegmatis mc2 155 and overexpression strains were diluted to O.D.595 nm = 0.05 in Middlebrook7H9 broth and incubated at 37°C with continuous shaking. The cultures were grown for 40 h, and the O.D.595nm was measured at every 2 h interval. The figure shows the mean O.D.595nmvalues obtained in three independent experiments. Bar represents the SD.

Mentions: From the experiments described so far, it is evident that the mutants lacking the basic residues in the CTD are compromised only for strand passage but are competent in site-specific DNA binding, cleavage and religation. Indeed, DNA relaxation activity of the WT enzyme was suppressed in the presence of the deletant enzyme in vitro, indicating the competition between the two proteins for the same substrate DNA (Supplementary Figure S5). Hence, in principle, they should exert dominant negative effect on enzyme function when expressed in vivo. To understand the consequence of the deletions on intracellular topoI function, MstopoI ΔB23 was overexpressed in M. smegmatis, and the growth was monitored. Interestingly, the cells overexpressing the deletant topoI showed significant reduction in growth compared with the cells overexpressing the WT enzyme. The cells expressing the deletant showed, increased lag period (16 h) compared with WT (12 h), 1.6-fold reduced growth rate and were affected in attaining maximum growth density (Figure 6). The results suggest that the overexpression of a functionally compromised form of the enzyme can compete out the WT enzyme for DNA binding, affecting the DNA relaxation activity in vivo leading to the reduced growth. This dominant negative effect thus establishes the importance of the crucial residues of CTD in completion of the DNA relaxation cycle.Figure 6.


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)

Overexpression of the deletant enzyme results in growth defect. Exponential phase cultures of M. smegmatis mc2 155 and overexpression strains were diluted to O.D.595 nm = 0.05 in Middlebrook7H9 broth and incubated at 37°C with continuous shaking. The cultures were grown for 40 h, and the O.D.595nm was measured at every 2 h interval. The figure shows the mean O.D.595nmvalues obtained in three independent experiments. Bar represents the SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt506-F6: Overexpression of the deletant enzyme results in growth defect. Exponential phase cultures of M. smegmatis mc2 155 and overexpression strains were diluted to O.D.595 nm = 0.05 in Middlebrook7H9 broth and incubated at 37°C with continuous shaking. The cultures were grown for 40 h, and the O.D.595nm was measured at every 2 h interval. The figure shows the mean O.D.595nmvalues obtained in three independent experiments. Bar represents the SD.
Mentions: From the experiments described so far, it is evident that the mutants lacking the basic residues in the CTD are compromised only for strand passage but are competent in site-specific DNA binding, cleavage and religation. Indeed, DNA relaxation activity of the WT enzyme was suppressed in the presence of the deletant enzyme in vitro, indicating the competition between the two proteins for the same substrate DNA (Supplementary Figure S5). Hence, in principle, they should exert dominant negative effect on enzyme function when expressed in vivo. To understand the consequence of the deletions on intracellular topoI function, MstopoI ΔB23 was overexpressed in M. smegmatis, and the growth was monitored. Interestingly, the cells overexpressing the deletant topoI showed significant reduction in growth compared with the cells overexpressing the WT enzyme. The cells expressing the deletant showed, increased lag period (16 h) compared with WT (12 h), 1.6-fold reduced growth rate and were affected in attaining maximum growth density (Figure 6). The results suggest that the overexpression of a functionally compromised form of the enzyme can compete out the WT enzyme for DNA binding, affecting the DNA relaxation activity in vivo leading to the reduced growth. This dominant negative effect thus establishes the importance of the crucial residues of CTD in completion of the DNA relaxation cycle.Figure 6.

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