Elucidation of an essential function of the unique charged domain of Plasmodium topoisomerase III

Topoisomerase III (TopoIII) along with RecQ helicases are required for the resolution of abnormal DNA structures that result from the stalling of replication forks. Sequence analyses have identified a putative TopoIII in the Plasmodium falciparum genome (PfTopoIII). PfTopoIII shows dual nuclear and mitochondrial localization. The expression and association of PfTopoIII with mtDNA are tightly linked to the asexual replication of the parasite. In this study, we observed that PfTopoIII physically interacts with PfBlm and PfWrn. Sequence alignment and domain analyses have revealed that it contains a unique positively charged region, spanning 85 amino acids, within domain II. A molecular dynamics simulation study revealed that this unstructured domain communicates with DNA and attains a thermodynamically stable state upon DNA binding. Here, we found that the association between PfTopoIII and the mitochondrial genome is negatively affected by the absence of the charged domain. Our study shows that PfTOPOIII can completely rescue the slow growth phenotype of the ΔtopoIII strain in Saccharomyces cerevisiae, but neither PfY421FtopoIII (catalytic-active site mutant) nor Pf(Δ259–337)topoIII (charged region deletion mutant) can functionally complement ScTOPOIII. Hydroxyurea (HU) led to stalling of the replication fork during the S phase, caused moderate toxicity to the growth of P. falciparum, and was associated with concomitant transcriptional up-regulation of PfTOPOIII. In addition, ectopic expression of PfTOPOIII reversed HU-induced toxicity. Interestingly, the expression of Pf(Δ259–337)topoIII failed to reverse HU-mediated toxicity. Taken together, our results establish the importance of TopoIII during Plasmodium replication and emphasize the essential requirement of the charged domain in PfTopoIII function.

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

Loss of TOP3B leads to increased R-loop formation and genome instability

Topoisomerase III beta (TOP3B) is one of the least understood members of the topoisomerase family of proteins and remains enigmatic. Our recent data shed light on the function and relevance of TOP3B to disease. A homozygous deletion for the TOP3B gene was identified in a patient with bilateral renal cancer. Analyses in both patient and modelled human cells show the disruption of TOP3B causes genome instability with a rise in DNA damage and chromosome bridging (mis-segregation). The primary molecular defect underlying this pathology is a significant increase in R-loop formation. Our data show that TOP3B is necessary to prevent the accumulation of excessive R-loops and identify TOP3B as a putative cancer gene, and support recent data showing that R-loops are involved in cancer aetiology.

Crystal structures of Sulfolobus solfataricus topoisomerase III reveal that its C-terminal novel zinc finger part is a unique decatenation domain

DNA topoisomerases are essential enzymes for a variety of cellular processes involved in DNA transactions. Many of the mechanistic insights into type IA DNA topoisomerases have principally come from studies on the prokaryotes and eukaryotes. However, a structural understanding of type IA topoisomerases in the Archaeal is lacking. Here we report the crystal structures of full-length Sulfolobus solfataricus topoisomerase III (Sso topo III) both by itself and in complex with an 8-base single-stranded DNA fragment, which were determined at 2.1 Å and 2.5 Å, respectively. The structures show that, as a member of type IA topoisomerases, Sso topo III adopts a torus-like architecture consisting of a four-domain core region and a novel C-terminal zinc finger domain (domain V). Upon binding to ssDNA, Sso topo III undergoes dramatic conformational changes, similar to those of other type IA topoisomerases. Structural analyses and biochemical assays revealed that domain V is essential for the DNA decatenation activity of Sso topo III. These findings establish Sso topo III as an alternative prototype of type IA topoisomerases to further understand the loop-independent decatenation mechanism in the enzyme-bridged strand passage model.

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

ABSTRACTThe role of DNA topoisomerase III (Topo III) in bacterial cells has proven elusive. Whereas eukaryotic Top IIIα homologs are clearly involved with homologs of the bacterial DNA helicase RecQ in unraveling double Holliday junctions, preventing crossover exchange of genetic information at unscheduled recombination intermediates, and Top IIIβ homologs have been shown to be involved in regulation of various mRNAs involved in neuronal function, there is little evidence for similar reactions in bacteria. Instead, most data point to Topo III playing a role supplemental to that of topoisomerase IV in unlinking daughter chromosomes during DNA replication. In support of this model, we show thatEscherichia coliTopo III associates with the replication forkin vivo(likely via interactions with the single-stranded DNA-binding protein and the β clamp-loading DnaX complex of the DNA polymerase III holoenzyme), that the DnaX complex stimulates the ability of Topo III to unlink both catenated and precatenated DNA rings, and that ΔtopBcells show delayed and disorganized nucleoid segregation compared to that of wild-type cells. These data argue that Topo III normally assists topoisomerase IV in chromosome decatenation by removing excess positive topological linkages at or near the replication fork as they are converted into precatenanes.IMPORTANCETopological entanglement between daughter chromosomes has to be reduced to exactly zero every time anE. colicell divides. The enzymatic agents that accomplish this task are the topoisomerases.E. colipossesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell’s genome, in order to do so.