Open circular DNA

Double-stranded DNA. It has previously been shown (19,21) that a mixture of linear, double-stranded DNA s of variable length forms an arc like one of the arcs in Figure 1, after fractionation by two-dimensional agarose gel electrophoresis. Because the sieving of open circular DNA increases more rapidly than the sieving of linear DNA as 2 increases (15,19), open circles variable in molecular weight form an arc closer to the first-dimension gel than the arc formed by linear DNA (19). However, unlike the arcs in Figure 1, the arcs for linear and circular double-stranded DNA must eventually become coincident as 6 increases, because these double-stranded DNA s all have the same po (15). 

The results of the previous paragraph indicate that a mixture of linear and open circular DNA s can be analysed to determine the amount and the mass distribution of each by use of a single two-dimensional agarose gel electrophoresis. However, if forms other than linear and open circular double-stranded DNA s are present, additional data are needed to complete such an analysis. The behavior of some branched DNA s has been determined (21). Additional studies of the behavior of branched, closed circular and single-stranded DNA s are needed to increase the capabilities of two-dimensional agarose gel electrophoresis for analysing mixtures of heterogeneous DNA s. 

Fig. 6 Photocleavage of supercoiled X174 DNA. d>X174 DNA (50 xM per base pair) was incubated with various compounds in 20% acetonitrile in Tris-HCl buffer (pH 7.5, 50 mM) at 25 °C for 2 h under a UV lamp (365 nm, 15 W) placed 10 cm from the sample, then analyzed by gel electrophoresis (0.9% agarose gel, ethidium bromide stain). Lane h DNA alone lane 2r. DNA following UV irradiation lanes 3-8-. compounds 14, 18, 8, 3, 15, and 16 (1000 p,M), respectively, following UV irradiation. Form 1 covalently closed supercoiled DNA Form II open circular DNA and Form 111 linear DNA...

Not so clear is the reason for a further peak (linear ds-DNA) between peak 1 and 2 observed at elevated temperatures or by means of in-phase a.c. polarography (d.m.e.) at room temperature/where it was formerly designated peak 2. With open-circular DNA, which shows similar adsorption properties to closed duplex DNA and linear ds-DNA, this peak 2 [however, named peak 2 in Reference should be present at room temperature (pH 8), too. 

DNA degradation by nucleases, a common problem in the assays of Mg-dependent topoisomerases, is not observed for these standard assay conditions. However, a substantial change in the electrophoretic mobility of supercoiled or open circular DNA is noted after incubation with early fractions, unless the samples are heated to 80° in the presence of SDS or treated with proteinase K. The aberrant migration of DNA is apparently caused by DNA-binding proteins. 

Fig. 4.14 Single molecular images of plasmid DNA extracted from form I (closed-circular DNA, (a)) and form II (open-circular DNA, (b)).

DNA catenanes prepared by the addition of T4 DNA ligase to a mixture of linear and nicked (open circular) DNAs. 

Fig. 1 Nucleic acid samples available for electrochemical experiments, (a, b) naturally occurring DNAs (bl) dsDNA fragments of defined lengths and nucleotide sequences can be conveniently prepared by cleavage with restriction endonucleases (c) NAs (both DNA and RNA) synthesized by enzymes (d) fully synthetic DNAs and RNAs of limited lengths (e) PCR, can amplify the desired DNA segment from template DNA. ds, double-stranded ss, single-stranded, kbp, kilobase pairs. Covalently closed circles of sc, supercoiled and rel, relaxed DNA. oc, open circular DNA (containing at least one interruption of the sugar-phosphate backbone) lin, linear DNA. See text for more details.

DNA ligases have been isolated from many sources, particularly from E.coli during phage infection (28). These enzymes can close breaks (not gaps) in the DNA phospho-diester chain. These enzymes can also transform an open circular DNA molecule into a closed circle by a covalent 3 -5 phosphodiester bond. Similarly, many cells possess "repair" enzymes that can repair gaps and defects in the DNA induced by various agents. Thus, Thy dimers formed by UV irradiation of DNA are hydrolyzed by specific DNases and are replaced by correct sequences by the repair enzymes. It is probable that this is the main role of the DNA polymerase of Kornberg. 

An assay that selectively measures endonucleolytic (but not exonucleolytic) action uses ccc DNA (ColEl plasmid) which contains ribonucleotides. The open-circular DNA product can be conveniently analyzed by agarose gel electrophoresis (12). RNase H activity can also be detected by in situ gel activity analysis (13). 

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