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Specialized DNA Structures

Specialized DNA structures are formed during the process of replication

The process of semiconservative replication illustrated in Fig. 5.3 requires that the two strands of the parental DNA duplex separate, after which they serve as a template for new DNA synthesis. Indeed, this allows the same base-pairing rules and hydrogen-bonding patterns to direct the order of nucleotides on the new DNA strand and to hold the two strands of duplex DNA together. The region of replicating DNA at which the two strands of the parental DNA are separated and two new daughter DNA molecules are made, each with one parental strand and one newly synthesized strand, is called a replication fork. Once DNA synthesis has initiated, elongation of the growing new DNA strand proceeds via the apparent movement of one or two replication forks. The replication fork(s) are at one or both ends of a distinct replicative structure called a replication eye or bubble, whichcan be visualized experimentally (Fig. 5.4a). Examination of replicating DNA molecules in the electron microscope shows regions where a single DNA duplex separates into two duplexes (containing newly synthesized DNA) followed by a return to a single duplex. This has the appearance of an eye or a bubble, and hence the structure is named accordingly. The replication bubble can result from either bidirectionalor unidirectionalreplication (Figure 5.4b). In bidirectional replication, two replication forks move in opposite directions from the origin, and hence each end of the bubble is a replication fork. In unidirectional replication, one replication fork moves in one direction from the origin. In this case, one end of a replication bubble is a replication fork and the other end is the origin of replication. If the chromosome is circular, the replication bubble makes a qstructure. As replication proceeds, the emergent daughter molecules (composed of one old strand and one new strand of DNA) are the identical to each other, ever increasing size, whereas the unreplicated portion of the chromosomes becomes smaller and smaller.

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Figure 5.4. Replication bubbles. Panel A shows diagrams of the replication bubbles or “eyes” that form when the two parental template chains are separated and copied during replication. Replication bubbles in a circular DNA molecule resemble the Greek letter theta, or q. Panel B shows electron micrographs of replicating polyoma virus DNA. The viral DNA from polyoma is duplex, circular and relatively small (about 5000 bp), which facilitates resolution of the parts of the replicating molecules. Each molecule in this panel shows two branch points, which are replication forks for polyoma, and three branches. Two of the branches in each molecule are the same length; these are the newly replicated portions of the DNA. The pictures are arranged to show progressively more replication. This is a copy of plate I from B. Hirt (1969) “Replicating Molecules of Polyoma Virus DNA”, Journal of Molecular Biology 40:141-144.Panel C illustrates that a replication bubble can result from either unidirectional or bidirectional replication. The origin of replication is labeled ori.

One could imagine making a new daughter DNA molecule via semiconservative replication by completely separating the two strands of a DNA molecule, and then using each separated strand as a template to make two daughter molecules that were separate during the entire process of replication. However, the visualization of replication bubbles during replication shows that the daughter DNA molecules are still connected to the parental molecule, producing the characteristic "eye" form (Fig. 5.4). Hence the separation of the two strands is localized to the replication fork. Although we discuss replication in terms of moving replication forks, it is more likely that the forks are stationary at a complex replication site, and the DNA is moved through this site rather than having the replication complex move along the DNA.

Although the replication bubbles, with two daughter duplexes being made at each replication fork, are commonly used in replicating cellular DNA, other types of replicative structure have been found. For example, a type of replicative structure used by some bacteriophage to quickly generate many copies of the viral DNA is the rolling circle(Figure 5.5). A rolling circle is a replicative structure in which one strand of a circular duplex is used as a template for multiple rounds of replication, generating many copies of that template. When replication proceeds by a rolling circle, replication of one strand of the duplex begins at a nick at the origin. The newly synthesized strand displaces the original nicked strand, which does not serve as a template for new synthesis. Thus the rolling circle mechanism copies only one strand of the DNA. Elongation proceeds by the replication machinery going around the template multiple times, in a pattern resembling a rolling circle. The large number of copies of a single strand of a phage genome made by the rolling circle are concatenated, or connected end-to-end. The single‑stranded DNA can be cleaved and ligated to generate unit length genomes, which are packaged into phage particles. This occurs in replication of single‑stranded DNA phages such as fX174 or M13. The DNA in the bacteriophage particle is single stranded, and this strand is called the viral or plus strand. After infection of a bacterial cell the viral DNA is converted to a duplex replicative form, which is the double-stranded form of viral DNA used in replication. The new strand of DNA made during the conversion of the infecting single-stranded DNA to the replicative form is, of course, complementary to the viral strand, and it serves as the template during replication by the rolling circle mechanism. Thus the many copies of DNA produced are the viral strand, and these are packaged into viral particles. The rolling circle mechanism is not restricted to single-stranded bacteriophage. In some bacteriophage, the displaced single strand is subsequently copied into a daughter DNA duplex. The concatenated, multiple copies of genome‑length duplex DNA produced in this way are then cleaved into genome‑sized molecules and packaged into viruses. Thus the rolling circle mechanism followed by copying of the displaced strand can also be used to replicate some double-stranded phage. This occurs in the second phase of replication of bacteriophage l.

   

Figure 5.5. Rolling circles are structures formed as replication intermediates for some bacteriophage.

 

To further illustrate the range of replicative structures, consider a third structure, which is observed during replication of mitochondrial DNA (Fig. 5.6). Mitochondrial DNA synthesis starts at a specific, unidirectional origin on one strand. Initially only one of the parental strands is used as template for synthesis of a new strand. This single new strand displaces the non-template parental strand, forming a displacement loop, or D loop. After replication of the first strand has proceeded about half way round the mitochondrial genome, synthesis of the other strand begins at a second origin and proceeds around the genome.

           

Figure 5.6.Displacement of D-loops in replication of mitochondrial DNA. Synthesis of one stand begins at an origin (ori1) and proceeds around the circular genome. The displaced strand is not immediately copied, and thus it forms a displacement loop, or D-loop. After synthesis from ori1has proceeded about halfway around the genome, synthesis of the other strand begins at a second origin (ori2). The thick gray arrow is oriented with the tip of the arrowhead at the 3’ end of the newly synthesized DNA.

Question 5.2.How does D loop synthesis differ from a replication eye?

Although this is not an exhaustive list, these are three examples illustrate a range of replicative structures. Now we turn to a more detailed examination of events that occur at the replication fork of a molecule in which replication is proceeding via replication bubbles.