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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

Strand passage mechanism of topoI. TopoI-mediated relaxation is proposed to occur by recognizing and binding to a single-stranded region within the supercoiled substrate (A and B), followed by nicking of the scissile strand. The enzyme undergoes conformational changes resulting in opening of the DNA gate (C). The complementary non-scissile strand held by the basic amino acid stretches in the CTD is passed through the opened DNA gate (D). Subsequently, two ends of the DNA are brought closer for religation (E) leading to the relaxation of supercoiled DNA. The enzyme releases the DNA or begins another catalytic cycle. Basic amino acid stretches are shown in cyan.
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gkt506-F7: Strand passage mechanism of topoI. TopoI-mediated relaxation is proposed to occur by recognizing and binding to a single-stranded region within the supercoiled substrate (A and B), followed by nicking of the scissile strand. The enzyme undergoes conformational changes resulting in opening of the DNA gate (C). The complementary non-scissile strand held by the basic amino acid stretches in the CTD is passed through the opened DNA gate (D). Subsequently, two ends of the DNA are brought closer for religation (E) leading to the relaxation of supercoiled DNA. The enzyme releases the DNA or begins another catalytic cycle. Basic amino acid stretches are shown in cyan.

Mentions: Based on the present findings, we propose a model for the mechanism of strand passage by MstopoI (Figure 7). According to this model, MstopoI binds scissile DNA in a site-specific manner by virtue of the NTD, whereas the CTD simultaneously interacts with the non-scissile DNA. On encountering the STS DNA region, the NTD cleaves the DNA to form the 5′-phosphotyrosine covalent adduct, followed by the opening of the DNA gate. Basic stretches in the CTD guide the non-scissile DNA from the central cavity to the DNA gate, leading to the strand passage, followed by resealing of the DNA ends, thus completing one cycle of DNA relaxation. After the catalysis, MstopoI may remain bound to the same DNA molecule through the basic amino acids, thus, enhancing the processivity of the enzyme.Figure 7.


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)

Strand passage mechanism of topoI. TopoI-mediated relaxation is proposed to occur by recognizing and binding to a single-stranded region within the supercoiled substrate (A and B), followed by nicking of the scissile strand. The enzyme undergoes conformational changes resulting in opening of the DNA gate (C). The complementary non-scissile strand held by the basic amino acid stretches in the CTD is passed through the opened DNA gate (D). Subsequently, two ends of the DNA are brought closer for religation (E) leading to the relaxation of supercoiled DNA. The enzyme releases the DNA or begins another catalytic cycle. Basic amino acid stretches are shown in cyan.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt506-F7: Strand passage mechanism of topoI. TopoI-mediated relaxation is proposed to occur by recognizing and binding to a single-stranded region within the supercoiled substrate (A and B), followed by nicking of the scissile strand. The enzyme undergoes conformational changes resulting in opening of the DNA gate (C). The complementary non-scissile strand held by the basic amino acid stretches in the CTD is passed through the opened DNA gate (D). Subsequently, two ends of the DNA are brought closer for religation (E) leading to the relaxation of supercoiled DNA. The enzyme releases the DNA or begins another catalytic cycle. Basic amino acid stretches are shown in cyan.
Mentions: Based on the present findings, we propose a model for the mechanism of strand passage by MstopoI (Figure 7). According to this model, MstopoI binds scissile DNA in a site-specific manner by virtue of the NTD, whereas the CTD simultaneously interacts with the non-scissile DNA. On encountering the STS DNA region, the NTD cleaves the DNA to form the 5′-phosphotyrosine covalent adduct, followed by the opening of the DNA gate. Basic stretches in the CTD guide the non-scissile DNA from the central cavity to the DNA gate, leading to the strand passage, followed by resealing of the DNA ends, thus completing one cycle of DNA relaxation. After the catalysis, MstopoI may remain bound to the same DNA molecule through the basic amino acids, thus, enhancing the processivity of the enzyme.Figure 7.

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