Nicked duplex

Fig. 5 Base pairing and base stacking free energy contributions to the helix thermal stability, as obtained from experiments with nicked duplexes, (a) Pairing shows negligible or even unfavorable effect on free energy, while stacking (b) gives favorable contribution with strong dependence on T, revealing both enthalpic and entropic effects. Adapted with permission from [9]...

Fig. 3.9. Diagram showing the principle of the nick-translation sequencing procedure. For the purpose of illustration the single unique product generated from one nicked molecule is shown. With a heterogeneous set of nicked duplexes nick-translation will proceed from each gap until terminated by the incorporation of a dideoxynucleotide. Since the corresponding deoxynucleotide is also incorporated in competition with the dideoxynucleotide, the eifect of non-random cleavage with DNAase I is minimized.

A displaced single-strand end of the nicked duplex. A, invades the homologous region of the unbroken duplex, B ... 

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). 

Continued branch migration yields a circular duplex with a nick and a displaced linear strand (left) or a partially single-stranded linear duplex (right). 

Figure 27-14 Schematic representation of DNA polymerase action on a nicked strand of DNA in which the nick has been enlarged. At the catalytic center new nucleotide units are added at the 3 end of a growing strand. At the 3 -5 exonuclease site the 3 terminal nucleotide may be removed hydrolytically. This will happen to the greatest extent if the nucleotide is poorly paired in the duplex. At the 5 -3 exonuclease site nucleotides are hydrolytically removed from the 5 end of a strand in the chain.265,267...

In the second and third stages of replication ( )X174 RF molecules are themselves replicated and are then used for synthesis of new viral (+) strands. At both stages a virally encoded gene A protein, which has endonuclease activity, nicks the duplex. Cutting the (+) strand it leaves a free 3-OH on DNA residue 4305, while the 5 -phospho group of residue 4306 becomes... 

The cross-stranded structure shown in Fig. 27-22 can be formed with all base pairs in both duplexes intact.526 527 All that is required is formation of a nick in each of the two polynucleotide chains and a rejoining of the backbones across the close gap between the duplexes. This model also accounts for the cutting of the two crossed strands at exactly equivalent points to terminate the process. Various mechanisms of recombination exist, and most make use of the key... 

The E. coli chromosome exists as a circular, folded, supercoiled duplex (a). This can be converted to a partially unfolded structure by brief treatment with RNase (c). There are 50-100 loops in the structure supercoiling may be selectively eliminated from individual loops by single-strand nicking of the DNA within the loop (b). 

The biochemical process of recombination occurs by breaking and rejoining DNA strands. The key reaction is strand displacement initiated at a nick in the chromosome. Then a protein called RecA (which stands for recombination rec bacteria are unable to recombine their DNA information and therefore are abnormally sensitive to UV radiation) binds to a single-stranded DNA fragment and catalyzer its exchange with the same sequence of the duplex. RecA protein is a strand displacement protein. See Figure 8-16. 

Free 3 -hydroxyl ends may occur naturally in double stranded DNA or as a result of deliberate nicking (i.e. single stranded breakage) of the molecule with endonucleases such as DNAase I (Figs. 1.2. and 1.3.). Such nicks are usually found to be randomly distributed along a duplex DNA. In a linear duplex the presence of nicks has little effect on the physicochemical properties of the molecule and for that reason they are often referred to as hidden breaks. To discover whether a linear duplex DNA is nicked or not... 

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