Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

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Skipping and sliding to optimize target search on protein-bound DNA and RNA

10.1101/2020.06.04.133629 ◽

2020 ◽

Author(s):

Misha Klein ◽

Tao Ju Cui ◽

Ian MacRae ◽

Chirlmin Joo ◽

Martin Depken

Keyword(s):

Nucleic Acid ◽

Single Molecule ◽

Diffusion Limit ◽

Optimal Search ◽

Dna And Rna ◽

Argonaute Proteins ◽

Large Pool ◽

Search Speed ◽

Crowded Environments ◽

Acid Substrate

Rapidly finding a specific nucleic-acid sequences in a large pool of competing off-targets is a fundamental challenge overcome by all living systems. To optimize the search and beat the diffusion limit, it is known that searchers should spend time sliding along the nucleic-acid substrate. Still, such sliding generally has to contend with high levels of molecular crowding on the substrate, and it remains unclear what effect this has on optimal search strategies. Using mechanistic modelling informed by single-molecule data, we show how sliding combined with correlated short-ranged skips allow searchers to maintain search speed on densely crowded substrates. We determine the conditions of optimal search, which show that an optimized searchers always spend more than half its time skipping and sliding along the substrate. Applying our theory to single-molecule data, we determine that both human and bacterial Argonaute proteins alternate between sliding 10 nt and skipping 30 nt along the substrate. We show that this combination of skipping and sliding lengths allows the searcher to maintain search speeds largely unaffected by molecular roadblocks covering up to 70% of the substrate. Our novel combination of experimental and theoretical approach could also help elucidate how other systems ensure rapid search in crowded environments.

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