Strand separation

Implicit in the functioning of the Watson-Crick DNA model is the idea that the strands of a DNA molecule must separate and new daughter strands must be synthesized in response to the sequence of bases in the mother strand. This is called semiconservative replication. Still, conservative replication, in which both strands of a daughter molecule are newly synthesized, could not be ruled out by consideration of the structure of DNA alone. 

This presented a more difficult problem How do the double-helical strands separate during DNA synthesis In a rapidly growing cell such as E. coli it has been calculated that if the strands separate by untwisting, the molecule would have to rotate at 10,000 rpm, a rate that is highly improbable. The answer to this problem lies in an understanding of the mechanism of DNA replication at the enzyme level. We will return to this subject after first considering the enzymes involved in DNA synthesis. 

1 30 cross-linked, 8% acrylamide gel often gives good strand separation while for longer DNA fragments of several hundred nucleotides heat denaturation in dimethylsulphoxide followed by electrophoresis in a 1 60 cross-linked 5% gel is preferable (Maxam and Gilbert, 1980). A suitable procedure is described in Procedure E (Experimental procedures). 

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PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

In these p-helix structures the polypeptide chain is coiled into a wide helix, formed by p strands separated by loop regions. In the simplest form, the two-sheet p helix, each turn of the helix comprises two p strands and two loop regions (Figure 5.28). This structural unit is repeated three times in extracellular bacterial proteinases to form a right-handed coiled structure which comprises two adjacent three-stranded parallel p sheets with a hydrophobic core in between. 

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

Tumor Necrosis Factor. Figure 1 Homotrimeric structure of soluble TNF (51 kDa). TNF monomers (17kDa) in individual subunits fold into a jelly role pattern that is composed of anti-parallel (3 strands separated by intervening loop regions, which are represented by green, red, or blue color. 

As described in section 4.1, the DNA double helix must unwind to allow access ofthe polymerase enzymes to produce two new strands ofDNA. This is facilitated by DNA gyrase, the target of the quinolones. Some agents interfere with the unwinding of the chromosome by physical obstruction. These include the acridine dyes, of which the topical antiseptic proflavine is the most familiar, and the antimalarial acridine, mepacrine. They prevent strand separation by insertion (intercalation) between base pairs from each strand, but exhibit very poor selective toxicity. 

Stabilization of all DNAs against thermal strand separation (Fig. 6c). 

Fig. 45. (a) Introduction of M(phen)31 complex into DNA oligomers, (b). Steady-state emission spectra of modified oligonucleotides in 0.01 m sodium phosphate buffer, pH 7.0, 0.1 NaCl. Top Ru(phen)3+-modified 20-mer duplex (solid line), a 1 1 mixture of non-comple-mentary Ru(phen)3+- and Os(phen)g+-modified 20-mers (- -) and a 20-mer duplex containing Ru(phen)g+ and Ps(phen)3+groups on different strands separated by one base pair (---). Bottom 20-mer duplex with 5 -terminal Rufphen) (solid line), and Ru(phen)jf/Os(phen)3 -containing analog (---). Reproduced with permission from Ref. (157). Copyright 1998, American Chemical Society. 

The multiplication phase the aggregates fall apart to give single strands, which now act as matrices. Complementary copying occurs, accompanied by a number of copying errors. Matrix and replication strand separate. 

P. Doty, J. Marmur, J. Eigen, and C.E. Schildkraut, Strand separation and specific recombination in deoxyribonucleic acids physical chemical studies. Proc. Natl. Acad. Sci. 46, 461-476 (1960). 

The double helix can be denatured by heating (melting). Denatured DNA, like denatured protein, loses its structure, and the two strands separate. Melting of DNA is accompanied by an increase in the absorbance of UV light with a wavelength of 260 nm. This is termed hyperchromicity and can by used to observe DNA denaturation. DNA denaturation is reversible. When cooled under appropriate conditions, the two strands find each other, pair correctly, and reform the double helix. This is termed annealing. 

Repeat cycle of DNA strand separation (denaturation), annealing to primer, and synthesis of DNA complementary to the strand template. 

Amplification is achieved by repeated cycles (20 to 50) of heating (for strand separation) and cooling (for specific hybridization). Amplicons result upon ligation of adjacent probes. A commercialized automated system achieves detection... 

The two major types of nucleic acids are DNA and RNA. Nucleic acids are polyphosphate esters containing the phosphate, sugar, and base moieties. Nucleic acids contain one of five purine or pyrimidine bases that are coupled within double-stranded helices. DNA, which is an essential part of the cell s chromosome, contains the information for the synthesis of protein molecules. For double-stranded nucleic acids, as the two strands separate, they act as a template for the construction of a complementary chain. The reproduction or duplication of the DNA chains is called replication. The DNA undergoes semiconservative replication where each of the two new strands contains one of the original strands. 

Dissolve DNA strands separately in an appropriate followed by slow cooling to 5°C. 

Double helix—The shape of DNA molecules, discovered by James Watson and Francis Crick. The double helix is made up of two chains of DNA bound to each other by weak chemical bonds between pairs of complementary bases. This base pairing allows the DNA to be copied precisely when the strands separate as cells divide. 

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