Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

Szafran, Marcin Jan; Skut, Patrycja; Ditkowski, Bartosz; Ginda, Katarzyna; Chandra, Govind; Zakrzewska‐Czerwińska, Jolanta; Jakimowicz, Dagmara

doi:10.1128/jb.00798-13

Cited by 30 publications

(76 citation statements)

References 68 publications

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“…The sequence analysis and the bulk assays of Sc TopA activity, which suggested rapid supercoil removal [(21) and see also Figure 7A] prompted us to analyse the properties of Sc TopA in single-molecule experiments using magnetic tweezers. In these experiments, the DNA substrates are tethered between a glass surface and a 1-μm magnetic bead and manipulated as described in the Materials and Methods section (see Figure 1A and B).…”

Section: Resultsmentioning

confidence: 99%

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

Szafran¹,

Strick²,

Strzałka³

et al. 2014

Nucleic Acids Res

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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.

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Section: Resultsmentioning

confidence: 99%

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

Szafran¹,

Strick²,

Strzałka³

et al. 2014

Nucleic Acids Res

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“…Since mycobacterial HupB presumably interacts with topoisomerase I (TopA) (45), the lack of HupB may disturb the topological homeostasis of the chromosome. In Streptomyces, TopA is recruited to the ParB complexes during sporulation (57,58), which suggests that HupB may play a role in the segregation/organization of oriC regions. Thus, as we previously suggested, HupB may be involved in organizing newly replicated regions and (potentially) in coordinating chromosome replication with segregation.…”

Section: Discussionmentioning

confidence: 99%

The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB

et al. 2018

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The bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of the chromosome and that the HupB-binding sites exhibit a bias from the origin () to the terminus (). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track the and replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation in, the sister regions of move asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation. Although our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation in , the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.

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“…Mycobacterial topoisomerase I may have functional roles associated with topoisomerase III in bacteria that have this additional type IA topoisomerase. The actinobacterium Streptomyces coelicolor also does not have topoisomerase III, and it has been hypothesized that S. coelicolor topoisomerase I is recruited to ParB complexes because the topoisomerase activity is needed to resolve segregating chromosomes 44 .…”

Section: Discussionmentioning

confidence: 99%

Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein–Protein Interaction

Banda

Cao

Tse‐Dinh

2017

Journal of Molecular Biology

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We report here a distinct mechanism of interaction between topoisomerase I and RNA polymerase in Mycobacterium tuberculosis and Mycobacterium smegmatis that has evolved independently from the previously characterized interaction between bacterial topoisomerase I and RNA polymerase. Bacterial DNA topoisomerase I is responsible for preventing the hyper-negative supercoiling of genomic DNA. The association of topoisomerase I with RNA polymerase during transcription elongation could efficiently relieve transcription-driven negative supercoiling. Our results demonstrate a direct physical interaction between the C-terminal domains of topoisomerase I (TopoI-CTD) and the β′ subunit of RNA polymerase of M. smegmatis in the absence of DNA. The TopoI-CTD in mycobacteria are evolutionarily unrelated in amino acid sequence and three-dimensional structure to the TopoI-CTD found in the majority of bacterial species outside Actinobacteria, including Escherichia coli. The functional interaction between topoisomerase I and RNA polymerase has evolved independently in mycobacteria and E. coli, with distinctively different structural elements of TopoI-CTD utilized for this protein-protein interaction. Zinc ribbon motifs in E. coli TopoI-CTD are involved in the interaction with RNA polymerase. For M. smegmatis TopoI-CTD, a 27 amino acid tail that is rich in basic residues at the C-terminal end is responsible for the interaction with RNA polymerase. Overexpression of recombinant TopoI-CTD in M. smegmatis competed with the endogenous topoisomerase I for protein-protein interactions with RNA polymerase. The TopoI-CTD overexpression resulted in decreased survival following treatment with antibiotics and hydrogen peroxide, supporting the importance of the protein-protein interaction between topoisomerase I and RNA polymerase during stress response of mycobacteria.

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Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

Cited by 30 publications

References 68 publications

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

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

The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB

Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein–Protein Interaction

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