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

Ahmed, Wareed; Bhat, Anuradha Gopal; Leelaram, Majety Naga; Menon, Shruti; Nagaraja, Valakunja

doi:10.1093/nar/gkt506

Cited by 27 publications

(51 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Besides the basic C-terminal tail, there are two more stretches of basic amino acids. 13 Based on the prediction, one is a part of the insertion on the loop between the β3 and β4 strands of D6, and the other is a part of the linker between the D7 and D8 domains (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Deletions of the stretches of basic amino acids in the C-terminal region of MtTOP1 have been shown to affect DNA binding and DNA strand passage. 13 The deletions would unlikely make an impact on the folding of the individual C-terminal domains but may affect the relative orientations of the C-terminal domains and the inter-domain flexibility.…”

Section: Resultsmentioning

confidence: 99%

“…13 It was labeled at the 5′-end with γ- 32 P-ATP and T4 polynucleotide Kinase. Cleavage activity was assayed by incubating the labeled oligonucleotide (0.5 pmole) with MtTOP1 and MtTOP1-704t in 5 μl of 10 mM Tris-HCl pH 8.0 at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

Tan

Cao

Cheng

et al. 2016

Journal of Molecular Biology

View full text Add to dashboard Cite

The DNA Topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug resistant tuberculosis (TB). In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetra-cysteine motifs seen in the C-terminal domains of E. coli topoisomerase I. A construct (a.a. A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze ssDNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 Å resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in the other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel β-sheet stabilized by a crossing-over α-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

Tan

Cao

Cheng

et al. 2016

Journal of Molecular Biology

View full text Add to dashboard Cite

show abstract

“…However, while recombinant M. tuberculosis topoisomerase I restores nearly all of the relaxation activity, it did not alleviate the increased antibiotic sensitivity observed for strain DPB636 due to the topA66 CTD mutation. The CTD sequences of E. coli and M. tuberculosis topoisomerase I have diverged in evolution even though they appear to play similar roles of interaction with a passing strand of DNA in the mechanism of relaxation of negative DNA supercoiling (33,34). This suggests that the protein-protein interaction between the CTD of topoisomerase I and RNA polymerase (19) may be the basis for the increased bacteriostatic and bactericidal effect of antimicrobials observed for the topA66 mutant.…”

Section: Discussionmentioning

confidence: 99%

Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation

Yang

Annamalai

Cheng

et al. 2015

Antimicrob Agents Chemother

View full text Add to dashboard Cite

c Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcriptiondriven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response. Microbial pathogens resistant to current antibiotics are becoming an increasingly urgent public health crisis. For example, quinolones are widely used broad-spectrum antimicrobial agents that are highly effective for rapid bactericidal outcomes (1), but quinolone resistance can be acquired rapidly by the bacterial pathogens via a number of mechanisms (2, 3). The bacterial SOS response system has been shown to contribute to the increase in antibiotic resistance (4). There are many factors that can potentially lead to the increased resistance in response to the SOS-inducing antimicrobials. These factors include elevations in the expression levels of error-prone DNA polymerases (5) or plasmidmediated antibiotic resistance determinants (6, 7), higher rates of transfer of resistance determinants (8, 9), and increases in the numbers of persistors in the bacterial population (10).The transcriptome in the bacterial cell must adjust rapidly to the antibiotic challenge for survival. Due to resistance to the rotational motion of the transcription ensemble around the DNA during transcription, the transcription loci can accumulate positive supercoils ahead of the RNA polymerase (RNAP)-nascent RNA complex and negative supercoils behind the comple...

show abstract

“…Thus, increased enzyme processivity is presumably linked to the C-terminal stretch of basic amino acids as indicated by Ahmed et al. (37). Predictions of secondary structure suggest that the C-terminus of Sc TopA forms an α-helix with basic amino acids precisely oriented in a helical fashion on the surface of the α-helix and which may constitute the domain interacting with the highly compacted supercoiled DNA, providing higher affinity to substrate.…”

Section: Discussionmentioning

confidence: 99%

A highly processive topoisomerase I: studies at the single-molecule level

Szafran¹,

Strick²,

Strzałka³

et al. 2014

Nucleic Acids Res

View full text Add to dashboard Cite

Amongst enzymes which relieve torsional strain and maintain chromosome supercoiling, type IA topoisomerases share a strand-passage mechanism that involves transient nicking and re-joining of a single deoxyribonucleic acid (DNA) strand. In contrast to many bacterial species that possess two type IA topoisomerases (TopA and TopB), Actinobacteria possess only TopA, and unlike its homologues this topoisomerase has a unique C-terminal domain that lacks the Zn-finger motifs characteristic of type IA enzymes. To better understand how this unique C-terminal domain affects the enzyme's activity, we have examined DNA relaxation by actinobacterial TopA from Streptomyces coelicolor (ScTopA) using real-time single-molecule experiments. These studies reveal extremely high processivity of ScTopA not described previously for any other topoisomerase of type I. Moreover, we also demonstrate that enzyme processivity varies in a torque-dependent manner. Based on the analysis of the C-terminally truncated ScTopA mutants, we propose that high processivity of the enzyme is associated with the presence of a stretch of positively charged amino acids in its C-terminal region.

show abstract

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

Cited by 27 publications

References 46 publications

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation

A highly processive topoisomerase I: studies at the single-molecule level

Contact Info

Product

Resources

About