Interaction with single‐stranded DNA‐binding protein localizes ribonuclease HI to DNA replication forks and facilitates R‐loop removal

Wolak, Christine; Jiang, Hui; Soubry, Nicolas; Sandler, Steven J.; Reyes‐Lamothe, Rodrigo; Keck, James L.

doi:10.1111/mmi.14529

Cited by 18 publications

(23 citation statements)

References 112 publications

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“…3 G and 5 A ), raising the possibility of yet undiscovered roles for SSB in RNA metabolism. Indeed, recently SSB was found to enhance the function of RNase HI in R-loop removal ( 56 ).…”

Section: Resultsmentioning

confidence: 99%

Phase separation by ssDNA binding protein controlled via protein−protein and protein−DNA interactions

Harami

Kovács

Pancsa

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

106

165

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Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein−protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid−liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

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“…3 G and 5 A ), raising the possibility of yet undiscovered roles for SSB in RNA metabolism. Indeed, recently SSB was found to enhance the function of RNase HI in R-loop removal ( 56 ).…”

Section: Resultsmentioning

confidence: 99%

Phase separation by ssDNA binding protein controlled via protein−protein and protein−DNA interactions

Harami

Kovács

Pancsa

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

106

165

View full text Add to dashboard Cite

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“…Since the chromosomally-located reporters gyrB::lacZ and topA::lacZ , showed wild-type levels of topoisomerase activity in Δ rnhC p rnhC , but only report on the topology status of the chromosome, we concluded that the increased number of R-loops detected in the complemented ΔrnhC strain must therefore be plasmid-borne. It is possible that increased hyper-negative supercoiling of the plasmid, either through localised transcription effects (48) and/or titration of RNase HI activity to the chromosome (49), may have affected binding efficiencies of some of the proteins involved in plasmid replication, resulting in increased initiation frequencies, as well as promotion of R-loop formation.…”

Section: Resultsmentioning

confidence: 99%

RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Al-Zubaidi

Cheung

et al. 2021

Preprint

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Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

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“…Recently, Arabidopsis OSB1 was reported to function together with ribonuclease (RNase) H1 in decreasing the formation of R‐loops in mitochondria (Cheng et al, 2021). In E. coli , the interaction of SSB and RNase HI localizes RNase HI to DNA replication sites and facilitates the removal of R‐loops that block DNA replication (Wolak et al, 2020). Considering that OSB1 is also an ssDNA‐binding protein, we propose that SSB1, alone or with SSB2, competes with OSB1 to bind to ssDNA at R‐loop sites to balance mtDNA replication and mtDNA HR surveillance (Figure S4).…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis mitochondrial single‐stranded DNA‐binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Qian

Zheng

Wang

et al. 2022

JIPB

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Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants.Here, we show that the two mitochondriontargeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two-to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/ 2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.

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Interaction with single‐stranded DNA‐binding protein localizes ribonuclease HI to DNA replication forks and facilitates R‐loop removal

Cited by 18 publications

References 112 publications

Phase separation by ssDNA binding protein controlled via protein−protein and protein−DNA interactions

Phase separation by ssDNA binding protein controlled via protein−protein and protein−DNA interactions

RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Arabidopsis mitochondrial single‐stranded DNA‐binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

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