Nicked DNA

Template specificity Nicked DNA template, RNA primer Nicked DNA template, DNA primer Ribonucleotide template and DNA primer... 

Photolysis of [Co(cyclam)(H20)(CH3)]2+ is known to produce methyl radicals via Co-C bond homolysis (235). The complex is stable in water and in the presence of oxygen, and it is capable of converting su-percoiled plasmid DNA into a nicked circular form after 2 h in ordinary room light (236). Longer photolysis results in increased yields of nicked DNA. The complex is not selective in its attack on nucleotides. 

Blanche et al. [45] showed that the P-CAC technology is very promising for the purification of Plasmid DNA at preparative scale especially when resins with low binding capacities for the product of interest are used. The aim of the study was to purify the Plasmid DNA out of a clear lysate of E. coli. The lysate containing RNA, nicked DNA, as well as the Plasmid DNA was loaded onto the annular column filled with Poros 20 R2 beads as the stationary phase. The chromatographic process for the purification is shown in Fig. 7. 

The feed is introduced at the top of the annular column at the 0° position. The feed solution is followed by a wash buffer, which is introduced to the annular column through the main inlet port. A 1 vol.% mixture of 2-propanol in a 100 mmol/1 ammonium acetate buffer was used as wash buffer. In the washing zone the nicked DNA followed by the RNA are eluted from the column according to their affinity to the resin. At 180° offset from the feed nozzle the elution buffer (5 vol.%) 2-propanol in 100 mmol/1 ammonium acetate) was pumped to the annulus of the column. The elution buffer was used to strip off the bounded Plasmid DNA. Regeneration of the column was achieved by a 20 vol.% mixture of 2-propanol in 100 mmol/1 ammonium acetate buffer. All of the above-mentioned steps, i. e., feed, wash, elution, and regeneration, were done simultaneously and continuously on the P-CAC system. 

Figure 8 compiles some pertinent analytical data of the obtained fractions. Most of the nicked DNA (chromatogram C in Fig. 8 pooled nicked DNA fraction from the wash zone of the P-CAC) as well as the RNA (chromatogram D in Fig. 8 pooled RNA fraction from the wash zone of the P-CAC) were removed in the wash zone. Chromatogram A in Fig. 8 represents the composition of the P-CAC feedstock. Chromatogram B in Fig. 8 demonstrates the purity of the pooled plasmid DNA fraction obtained with the P-CAC. 

Plasmid DNA (pUC18) Fab fragment from an IgG purified from human sera pH 7.5,30°C Nicked DNA Autoantibodies Human serum IgG fraction (Fab) was found to hydrolyse DNA. Unknown immunogen 4.3 x 101 1.4 x 101 nr 6.5... 

Langst G, Becker PB (2001) ISWl induces nucleosome sliding on nicked DNA. Mol Cell 8 1085—1092 Langst G, Becker PB (2004) Nucleosome remodeling one mechanism, many phenomena Biochim Biophys Acta 1677 58-63... 

Suck, D., Lahm, A. and Oefner, C. (1988). Structure refined to 2 A of a nicked DNA octanucleotide complex with DNase I. Nature 332,464r-468. 

DNA ligases. These enzymes, which are essential to replication, have a specific function of repairing "nicked" DNA 261 304-307a Such DNA, as indicated in... 

DNA strand breakage is caused by EDTA-Cu2+ in the presence of a thiol (Mukherjee and Chatterjee 1995). In this study it has, however, also been reported that Cu2+ (in the presence of H202 and Cl-) nick DNA in the absence of a deliberately added reductant. The latter observation has been corroborated by Yamamoto and Kawanishi (1989), and it has been suggested that H202 can serve as a reductant under these conditions. The pattern of DNA fragments in... 

Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, J., and Pelletier, H. (1997). Crystal structures of human DNA polymerase [S complexed with gapped and nicked DNA Evidence for an induced fit mechanism. Biochemistry 36, 11205-11215. 

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze transfer ofmultiple ADP-ribose units onto target proteins, as shown in Figure 8.6. They are DNA-binding proteins with a zinc-finger motif and require nicked DNA (with single- or double-strand breaks) for activity. They are present in the ceU in high concentrations about one molecule of enzyme for each kilobase of DNA (Hayaishi and Ueda, 1977 D Amours et al., 1999). 

Addition of chloroquine or ethidium bromide in the running buffer resolves nicked DNA vs relaxed DNA (21). 

Fig. 3. (A) Relaxation of SV40 DNA by purified topi. Lane 1, DNA alone. The majority of the DNA is in its supercoiled form, but a fraction of the DNA is nicked. Lane 2 SV40 + topi. Relaxed DNA appears as multiple bands corresponding to the different changes in linking numbers between the supercoiled and the nicked DNA. (B) Decatenation of kDNA by top2. Lane 1, DNA alone lane 2 and 3 kDNA + top2 for 15 min and 30 min incubation, respectively. Decatenation is measured by the disappearance of the band in the well and appearance of lower bands (indicated by arrows).

Separation between relaxed and nicked DNA is possible using agarose gels containing 2 pg/mL chloroquine (21). 

First, it is found that in nicked DNA E. coli topoisomerase I binds close to the site of the nick and, when allowed to cleave the DNA, cuts the intact strand nearly opposite the nick. Second, an important feature of the catenation reaction is that at least one DNA partner must contain a nick or a single-stranded gap. If the enzyme binds at the nick and allows passage of DNA through the intact strand, one can conclude that the... 

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