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Two convergent pathways of DNA knotting in replicating DNA molecules as revealed by ?-curve analysis.

ABSTRACT: During DNA replication in living cells some DNA knots are inadvertently produced by DNA topoisomerases facilitating progression of replication forks. The types of DNA knots formed are conditioned by the 3D organization of replicating DNA molecules. Therefore, by characterizing formed DNA knots it is possible to infer the 3D arrangement of replicating DNA molecules. This topological inference method is highly developed for knotted DNA circles. However, partially replicated DNA molecules have the form of ?-curves. In this article, we use mathematical formalism of ?-curves to characterize the full possibilities of how knotting can occur during replication of DNA molecules in vivo. To do this, we reanalyze earlier experimental studies of knotted, partially replicated DNA molecules and the previously proposed pathway of their formation. We propose a general model of knotting in replication intermediates, and demonstrate that there is an additional, equally important, parallel knotting pathway that also explains how DNA topoisomerases can produce experimentally observed knotted ?-curves. Interestingly, both pathways require intertwining of freshly replicated sister duplexes (precatenanes).

SUBMITTER: O'Donnol D

PROVIDER: S-EPMC6158496 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Two convergent pathways of DNA knotting in replicating DNA molecules as revealed by θ-curve analysis.

O'Donnol Danielle D Stasiak Andrzej A Buck Dorothy D

Nucleic acids research 20180901 17

During DNA replication in living cells some DNA knots are inadvertently produced by DNA topoisomerases facilitating progression of replication forks. The types of DNA knots formed are conditioned by the 3D organization of replicating DNA molecules. Therefore, by characterizing formed DNA knots it is possible to infer the 3D arrangement of replicating DNA molecules. This topological inference method is highly developed for knotted DNA circles. However, partially replicated DNA molecules have the ...[more]

PMID: 29982678

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Project description:BackgroundMethylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler.MethodsWe first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample.ResultsFor validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues.ConclusionWe have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The technique does not depend on any operator manipulation or interpretation of the melt curves, and is suitable for use in any laboratory with a real-time thermocycler. The parameters derived provide an objective description and quantitation of the methylation in a specimen, and can be used to for statistical comparisons of methylation between specimens.

Dataset Information

Two convergent pathways of DNA knotting in replicating DNA molecules as revealed by ?-curve analysis.

Publications

Two convergent pathways of DNA knotting in replicating DNA molecules as revealed by θ-curve analysis.

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