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

Banda, Srikanth; Cao, Nan; Tse‐Dinh, Yuk‐Ching

doi:10.1016/j.jmb.2017.08.011

Cited by 19 publications

(19 citation statements)

References 51 publications

(56 reference statements)

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“…However, during transcription elongation, continuously generated negative supercoils must be resolved by the action of TopoI throughout gene body as seen in the present study. In this context, the observation that TopoI physically interacts with RNAP gains significance (61). The authors hypothesized that the enzyme may migrate with the transcription elongation complex to prevent the accumulation of supercoiling (61).…”

Section: Discussionmentioning

confidence: 99%

Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation

Rani

Nagaraja

2018

Nucleic Acids Research

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DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.

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Section: Discussionmentioning

confidence: 99%

Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation

Rani

Nagaraja

2018

Nucleic Acids Research

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“…coli [ 11 , 12 ] and this interaction is also found between M . tuberculosis topo I and RNAP, though in that case by a distinct mechanism [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli

et al. 2018

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Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.

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“…This indicates that topo I activity is always required during transcription, including when the cells are exposed to various stresses [43]. An interaction between RNAP and topo I, however involving different portions of the proteins as compared to E. coli, has also been observed for M. tuberculosis [44]. This may suggest that such an interaction has been evolutionarily conserved in bacteria.…”

Section: Viability Of Single Topa Null Mutants and The Role Of Topo Imentioning

confidence: 99%

Supercoiling, R-Loops, Replication and the Functions of Bacterial Type 1A Topoisomerases

Brochu

Breton

Drolet

2020

Genes

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Type 1A topoisomerases (topos) are the only topos that bind single-stranded DNA and the only ones found in all cells of the three domains of life. Two subfamilies, topo I and topo III, are present in bacteria. Topo I, found in all of them, relaxes negative supercoiling, while topo III acts as a decatenase in replication. However, recent results suggest that they can also act as back-up for each other. Because they are ubiquitous, type 1A enzymes are expected to be essential for cell viability. Single topA (topo I) and topB (topo III) null mutants of Escherichia coli are viable, but for topA only with compensatory mutations. Double topA topB null mutants were initially believed to be non-viable. However, in two independent studies, results of next generation sequencing (NGS) have recently shown that double topA topB null mutants of Bacillus subtilis and E. coli are viable when they carry parC parE gene amplifications. These genes encode the two subunits of topo IV, the main cellular decatenase. Here, we discuss the essential functions of bacterial type 1A topos in the context of this observation and new results showing their involvement in preventing unregulated replication from R-loops.

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

Cited by 19 publications

References 51 publications

Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation

Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli

Supercoiling, R-Loops, Replication and the Functions of Bacterial Type 1A Topoisomerases

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