Nick in DNA

DNase 1 j Under appropriate conditions, produces j single-stranded nicks in DNA. Nick translation mapping of hypersensitive sites mapping protein-DNA interactions. 

DNA polymerase and 5 - 3 exonuclease functions of the enzyme. Nucleotide hydrolysis in the 5 - 3 direction concomitant with nucleotide polymerization results in translocation of the position of the discontinuity by a process termed nick translation (Fig. 2) (30). Discontinuities or nicks in DNA can be introduced into intact DNA by limited digestion with pancreatic DNase I, which generates 3 -hydroxyl termini in double-stranded DNA. If radioactive nucleotides are used in the reaction with DNA polymerase 1, randomly and uniformly labeled DNA is produced (31). 

Both topi and top2 can remove supercoils by making transient nicks in DNA and allowing the passage of another single- or double-stranded DNA molecule through the nick. However, only top2 is able to catenate or decatenate closed duplex DNA. 

It is not known whether magnesium pyrophosphate and the 3 -hydroxyl group of a nick in DNA share a common acceptor binding site of DNA ligase, in analogy to the acceptors in reactions (35a), (36a), and (37a) discussed above. It seems unlikely that a limited binding site could acconunodate such diverse ac-... 

The uranyl(VI) ion (UO2 ) is known to uiduce single strand "nicks in DNA on... 

Next we studied DNA repair by the number of nicks in DNA of the liver after MAM acetate treatment. The length of single-stranded DNA was analyzed by agarose gel electrophoresis under alkaline conditions [6]. 

Bleomycins are a family of structurally related compounds produced by Streptomyces verticillus (Umezawa et al. 1966). They act through a peculiar mechanism, causing nicks in DNA strands by a complex reaction involving oxygen and metals. A mixture of bleomycins A2 and B2 was introduced in clinics in 1966 for the treatment of squamous cell carcinomas and malignant lymphomas and is still used, mainly in combinations. 

Ligation Reaction. The nick in the phosphate backbone is repaired by DNA ligase. A similar excision repair mechanism exists in mammalian cells (see, e.g., Cleaver, 1983). 

A related observation is that fully relaxed supercoiled DNA/dye complexes are somehow different from nicked circular DNA/dye complexes in the presence of the same concentration of free dye, where the binding ratios should be the same. This is readily seen in gel electrophoresis in the presence of sufficient dye concentration so that at least one, but not all, of the topoisomers is positively supercoiled. The slowest moving, presumably fully relaxed, topoisomer migrates significantly faster than the nicked circle, and this difference increases with the amount of dye present. This is not observed with chloroquine, perhaps because the effect is too small. However, it is readily apparent in the original gels of Keller0 61) in which ethidium was used to unwind the topoisomers. We have confirmed this effect for ethidium and have observed similar behavior for proflavine, 9-aminoacridine, and quinacrine. 

Following Dpn I digestion, the remaining DNA must be grown in sufficient amounts for further manipulation and so it is introduced into competent bacteria. Once introduced into competent cells, the nicks in the duplex DNA are repaired and the intact construct can be replicated by bacterial machinery. As described earlier, we typically make our own competent cells, but supercompetent cells are available from a number of suppliers (e.g., Strategene and Promega). 

One of the very early research tools that were used to study the nucleosomal state of active genes were the nucleases, DNase I and Micrococcal nuclease. With the development of protocols for the isolation of nuclei from cells, it was possible to add these reagents to probe the accessibility of DNA. DNase I makes single nicks in double stranded DNA and when the DNA is associated with histones within the nucleosome, the DNA is extensively protected. Those nicks that are observed are found to occur only after extensive digestion and are limited to the outside surface of the DNA in 10 base increments [7,8]. Weintraub and Groudine in 1976 [9] first used this nuclease and observed that when nuclei from chicken erythrocytes were treated with DNase I, the active /1-globin gene was preferentially... 

DNA-(apurinic or apyrimidinic site) lyase [EC 4.2.99.18, formerly EC 3.1.25.2] acts on the C-Q-P bond 3 to the apurinic or apyrimidinic site in DNA. This bond is broken by a /3-elimination reaction, leaving a 3 -terminal unsaturated sugar and a product with a terminal 5 -phosphate. Note that this nicking of the phosphodiester bond is a lyase-type reaction, not hydrolysis. 

Assuming the vector DNA and the DNA fragment to be cloned have been suitably prepared by one or another of the methods described earlier in this section, the two species are joined to one another by annealing their ends and sealing the nicks with DNA ligase (Figure 3.13). 

FIGURE 25-16 Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps (D and ( ) lead to activation of the 5 phosphate in the nick. An AMP group is transferred first to a Lys residue on the enzyme and then to the 5 phosphate in the nick. In step (3), the 3 -hydroxyl group attacks this phosphate and displaces AMP, producing a... 

Figure 14. Motifs of double crossover molecules. The top row contains the three parallel isomers of double crossover (DX) molecules, DPE, DPOW and DPON P in their name indicates their parallel structure. Arrowheads indicate 3 ends of strands. Strands drawn with the same thickness are related by the vertical dyad axis indicated in the plane of the paper. DPE contains crossovers separated by an even number (two) of half-turns of DNA, DPOx by an odd number in DPOW, the extra half turn is a major groove spacing, in DPON, it is a minor groove spacing. The middle row illustrates two other DX isomers, DAE, and DAO. The symmetry axis of DAE is normal to the page (and broken by the nick in the central strand) the symmetry axis of DAO is horizontal within the page in DAO, strands of opposite thickness are related by symmetry. DAE+J, in the second row, is a DAE molecule, in which an extra junction replaces the nick shown in DAE.

Figure 5-17 Electron micrograph of a six-noded knot made by the Tn3 resolvase which is involved in movement of the Tn3 transposon (Chapter 27) from one location to another within the genome. Putative six-noded knot DNA was isolated by electroelution from an agarose gel. The knots, which are nicked in one strand, were denatured to allow the nicked strand to slide away and leave a ssDNA knot. This was coated with E. coli rec A protein (Fig. 27-24) to greatly thicken the strand and to permit the sign of each node (designated in the tracing) to be seen. From Wasserman et a/.184...

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