2. Origins can be mapped by autoradiography and electrophoresis

12.2 Origins can be mapped by autoradiography and electrophoresis

Key terms defined in this section

Replication fork is the point at which strands of parental duplex DNA are separated so that replication can proceed.

Figure 12.1 Replicated DNA is seen as a replication eye flanked by nonreplicated DNA.

A molecule of DNA engaged in replication has two types of regions. Figure 12.1 shows that when replicating DNA is viewed by electron microscopy, the replicated region appears as an eye within the nonreplicated DNA. The nonreplicated region consists of the parental duplex; this opens into the replicated region where the two daughter duplexes have formed.

The point at which replication is occurring is called the replication fork (sometimes also known as the growing point). A replication fork moves sequentially along the DNA, from its starting point at the origin. Replication may be unidirectional or bidirectional. The type of event is determined by whether one or two replication forks set out from the origin. In unidirectional replication, one replication fork leaves the origin and proceeds along the DNA. In bidirectional replication, two replication forks are formed; they proceed away from the origin in opposite directions.

Figure 12.2 Replicons may be unidirectional or bidirectional, depending on whether one or two replication forks are formed at the origin.

The appearance of a replication eye does not distinguish between unidirectional and bidirectional replication. As depicted in Figure 12.2, the eye can represent either of two structures. If generated by unidirectional replication, the eye represents one fixed origin and one moving replication fork. If generated by bidirectional replication, the eye represents a pair of replication forks. In either case, the progress of replication expands the eye until ultimately it encompasses the whole replicon.

Figure 12.3 A replication eye forms a theta structure in circular DNA.

Figure 12.4 The replication eye becomes larger as the replication forks proceed along the replicon. Note that the "eye" becomes larger than the nonreplicated segment. The two sides of the eye can be defined because they are both the same length. Photograph kindly provided by Bernard Hirt.

When a replicon is circular, the presence of an eye forms the θ-structure drawn in Figure 12.3. The successive stages of replication of the circular DNA of polyoma virus are visualized by electron microscopy in Figure 12.4.

Whether a replicating eye has one or two replication forks can be determined in two ways. The choice of method depends on whether the DNA is a defined molecule or an unidentified region of a cellular genome.

With a defined linear molecule, we can use electron microscopy to measure the distance of each end of the eye from the end of the DNA. Then the positions of the ends of the eyes can be compared in molecules that have eyes of different sizes. If replication is unidirectional, only one of the ends will move; the other is the fixed origin. If replication is bidirectional, both will move; the origin is the point midway between them.

Figure 12.5 Different densities of radioactive labeling can be used to distinguish unidirectional and bidirectional replication.

With undefined regions of large genomes, two successive pulses of radioactivity can be used to label the movement of the replication forks. If one pulse has a more intense label than the other, they can be distinguished by the relative intensities of labeling. These can be visualized by autoradiography. Figure 12.5 shows that unidirectional replication causes one type of label to be followed by the other at one end of the eye. Bidirectional replication produces a (symmetrical) pattern at both ends of the eye. This is the pattern usually observed in replicons of eukaryotic chromosomes (Huberman and Riggs, 1968).

Figure 12.6 The position of the origin and the number of replicating forks determine the shape of a replicating restriction fragment, which can be followed by its electrophoretic path (solid line). The dashed line shows the path for a linear DNA.

A more recent method for mapping origins with greater resolution takes advantage of the effects that changes in shape have upon electrophoretic migration of DNA. Figure 12.6 illustrates the two dimensional mapping technique, in which restriction fragments of replicating DNA are electrophoresed in a first dimension that separates by mass, and a second dimension where movement is determined more by shape. Different types of replicating molecules follow characteristic paths, measured by their deviation from the line that would be followed by a linear molecule of DNA that doubled in size.

A simple Y-structure, in which one fork moves along a linear fragment, follows a continuous path. An inflection point occurs when all three branches are the same length, and the structure therefore deviates most extensively from linear DNA. Analogous considerations determine the paths of double Y-structures or bubbles. An asymmetric bubble follows a discontinuous path, with a break at the point at which the bubble is converted to a Y-structure as one fork runs off the end.

Taken together, the various techniques for characterizing replicating DNA show that origins are most often used to initiate bidirectional replication. From this level of resolution, we must now proceed to the molecular level, to identify the cis-acting sequences that comprise the origin, and the trans-acting factors that recognize it.

Research

Huberman, J. and Riggs, A. D. (1968). On the mechanism of DNA replication in mammalian chromosomes. J. Mol. Biol. 32, 327-341.