Linear duplex molecules

A cut or nick is made in one of the two chains, and the circle rolls, peeling away one end of the cut chain, to yield the equivalent of two single chains, which function as templates for DNA synthesis at a fork. As the circle continues to roll, a linear duplex molecule containing multiple copies of the sequence is generated. The rolling-circle mechanism is also seen as a stage in the replication of some viral DNA molecules. 

Bacteriophage X is a genetically complex and extensively studied virus of E. coli. Because it has been the object of molecular genetic research, it was investigated and developed as a vector. The DNA of phage X, in the form in which it is isolated from the phage particle, is a linear duplex molecule of about 45.5 kb pairs. The entire DNA sequence has been determined (SI). At each end are short, single-stranded 5 projections of 12 nucleotides that are complementary in sequence and by which the DNA adopts a circular structure when it is injected into the host cell i.e., X DNA naturally has cohesive termini that associate to form the cos site. 

In living cells, DNA is found almost exclusively in the form of linear duplex molecules they may be supercoiled, stressed, or even knotted (White et al., 1987), but the helical axis is unbranched. DNA branched junction structures occur as intermediates in the process of recombination, but they are inherently unstable because of the twofold sequence symmetry, which occurs in natural DNAs. Oligonucleotides with stable branchings can, however, be made if the sequence symmetry is minimized. Solid-state synthesis (Chen and Seeman, 1991 Zhang and Seeman, 1992, 1994) then allows the construction of complex supramolecules with the 2-nm-thick DNA double strands. The procedure contains the following steps ... 

The detection of different conformations of DNA underscores the inherent flexibility built into the DNA duplex. All the conformations discussed thus far involve regular linear duplexes. Energetically favorable interactions with other molecules, particularly proteins, can induce additional conformations that do not result in major changes in either pair-... 

Linear duplex DNA can bind more ethidium bromide than covalently closed circular DNA of the same molecular weight. Why (Hint Ethidium bromide molecules bind between adjacent base pairs of DNA, causing the duplex to unwind in the region of binding.)... 

At some point, the torsional stress caused by the positive supercoils will become energetically unfavorable and the tendency of the molecule will be toward winding, thus preventing further binding of Et. Linear duplex DNA does not undergo this torsional stress because it is not covalently closed and would be expected to have a greater binding capacity for Et. 

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... 

Exonuclease III is a 3 - 5 exonuclease which is specific for double-stranded DNA (Richardson et al., 1964). Digestion of a linear duplex proceeds from the 3 -ends of both strands and the products of a complete digest are two single-stranded molecules, each about half the length of the original duplex with probably only a small amount of complementarity remaining between them at their 3 -ends. Provided the exonuclease is free from contaminant activities, these single... 

Consider a linear duplex DNA molecule that undergoes replication through five successive generations. What would be the proportion of the original DNA within the total DNA population ... 

Addition of Mg to these complexes leads to their dissociation. However, addition of alkali leads to the appearance of cleaved DNA molecules having protein covalently attached to the DNA (see below). This enzyme also forms complexes with relaxed duplex DNA but these complexes are not dissociated by the addition of Mg and cleavage of the DNA is not found when alkali is added (Liu and Wang, 1979). Complexes between procaryotic topoisomerase I and DNA can also be detected by their retention on nitrocellulose filters. Using this method the preferential binding of the enzyme to an internal single-strand break in a linear duplex DNA has been shown (Dean et al., 1982 Dean and Cozzarelli, 1985). 

It is now known that DNA can adopt a variety of shs ies besides linear duplexes. One exan le is fmnadon d bent h es. In H ire 11.27. the linear structure (AO) is a DNA 50-mei The bend in the second strucbire (A5) is due to the ins on of five unpaired adenines. This rather modest change in shape cannot be expected to re t in a dramatic change in the anisotropy decay. Howevo, the ditfo ent rotational properties of these two molecules could be seen in the H) anisotropy data (Figure 11.28). These data were analyzed in tenns of a detailed hydrodynamic modd and... 

Fig. 3. A, "Slipped" circle formation by denaturation and annealing of a permuted collection of DNA duplexes. Notice that in the permuted DNA molecules the circular order of genes is constant, but the ends are repetitious. However, different DNA duplexes may have different repetitious ends. Upon annealing, the single strand fragments at either end of the same linear duplex may contain complementary sequences, which can undergo further reannealing to produce a circular duplex molecule. In some cases the single-stranded regions at the ends of the linear duplex will not be complementary and so will not result in circular duplex structures. (From Thomas. 1967. J. Cell Physio ., 70 (Suppl. 1) 13-34.)...

Fig. 8 2D DNA arrays, (a) Two DX molecules tile the plane. Conventional DX molecule, A, and a DX + J molecule, B, are seen to tile the plane. The extra domain black circles) on B leads to stripes In the array. The molecules are 4 x 16 nm, so the stripes are 32 nm apart, as seen in the AFM image on the right, (b) Four DX molecules (A-D ) tile the plane. This arrangement is similar to (a), but there is only one DX + J molecule, D, so the stripes are separated by 64 nm, as seen on the right, (c) TX array. Two TX tiles, A and B, are connected by complementarity between their first and third double helical domains, resulting in spaces between the tiles. D is a linear duplex that fits in the yellow rows, and C is a TX re-phased by three nucleotide pairs, and the rephased version is labeled C it fits into the gray rows and extends a double helical domain beyond the AB plane in both directions, as shown on the right...

Nevertheless, the RNA World hypothesis would seem to answer most of the questions raised above how can an essentially linear molecule be autocatalytic, how can it synthesize proteins, and how can it replicate Autocatalysis can occur because RNA can adopt a wide range of secondary and tertiary structures that position RNA monomers into a preorganized sequence and link them together, it can apply the same flexibility to bind other small molecules and catalyse their polymerization, and it could form weakly interacting sense and antisense duplexes. The main problem is... 

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