As indicated in Figures 4-33 and 4-34, both parental DNA strands that are exposed by local unwinding at a replication fork are copied into a daughter strand. In theory, DNA replication from a single origin could involve one replication fork that moves in one direction. Alternatively, two replication forks might assemble at a single origin and then move in opposite directions, leading to bidirectional growth of both daughter strands. Several types of experiments, including the one shown in Figure 4-35, provided early evidence in support of bidirectional strand growth.
The general consensus is that all prokaryotic and eu-karyotic cells employ a bidirectional mechanism of DNA replication. In the case of SV40 DNA, replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks. These then move away from the SV40 origin in opposite directions with leading- and lagging-strand synthesis occurring at both forks. As shown in Figure 4-36, the left replication fork extends DNA synthesis in the leftward direction; similarly, the right replication fork extends DNA synthesis in the rightward direction.
Unlike SV40 DNA, eukaryotic chromosomal DNA molecules contain multiple replication origins separated by tens to hundreds of kilobases. A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associates with other proteins required to load cellular hexa-meric helicases composed of six homologous MCM proteins.
▲ EXPERIMENTAL FIGURE 4-35 Electron microscopy of replicating SV40 DNA indicates bidirectional growth of DNA strands from an origin. The replicating viral DNA from SV40-infected cells was cut by the restriction enzyme EcoRI, which recognizes one site in the circular DNA. Electron micrographs of treated samples showed a collection of cut molecules with increasingly longer replication "bubbles," whose centers are a constant distance from each end of the cut molecules. This finding is consistent with chain growth in two directions from a common origin located at the center of a bubble, as illustrated in the corresponding diagrams. [See G. C. Fareed et al., 1972, J. Virol. 10:484; photographs courtesy of N. P Salzman.]
Two opposed MCM helicases separate the parental strands at an origin, with RPA proteins binding to the resulting single-stranded DNA. Synthesis of primers and subsequent steps in replication of cellular DNA are thought to be analogous to those in SV40 DNA replication (see Figures 4-34 and 4-36).
Replication of cellular DNA and other events leading to proliferation of cells are tightly regulated, so that the appropriate numbers of cells constituting each tissue are produced during development and throughout the life of an organism. As in transcription of most genes, control of the initiation
Leading-strand primer synthesis
Lagging-strand primer synthesis
Lagging-strand primer synthesis
Unwinding step is the primary mechanism for regulating cellular DNA replication. Activation of MCM helicase activity, which is required to initiate cellular DNA replication, is regulated by specific protein kinases called S-phase cyclin-dependent kinases. Other cyclin-dependent kinases regulate additional aspects of cell proliferation, including the complex process of mitosis by which a eukaryotic cell divides into two daughter cells. We discuss the various regulatory mechanisms that determine the rate of cell division in Chapter 21.
M FIGURE 4-36 Bidirectional mechanism of DNA replication. The left replication fork here Is comparable to the replication fork diagrammed In Figure 4-34, which also shows proteins other than large T-antigen. (Top) Two large T-antigen hexameric helicases first bind at the replication origin in opposite orientations. Step D: Using energy provided from ATP hydrolysis, the helicases move in opposite directions, unwinding the parental DNA and generating single-strand templates that are bound by RPA proteins. Step 2 Primase-Pol a complexes synthesize short primers base-paired to each of the separated parental strands. Step B: PCNA-Rfc-Pol 8 complexes replace the primase-Pol a complexes and extend the short primers, generating the leading strands (dark green) at each replication fork. Step 4 The helicases further unwind the parental strands, and RPA proteins bind to the newly exposed single-strand regions. Step 15: PCNA-Rfc-Pol 8 complexes extend the leading strands further. Step Primase-Pol a complexes synthesize primers for lagging-strand synthesis at each replication fork. Step H: PCNA-Rfc-Pol 8 complexes displace the primase-Pol a complexes and extend the lagging-strand Okazaki fragments (light green), which eventually are ligated to the 5' ends of the leading strands. The position where ligation occurs is represented by a circle. Replication continues by further unwinding of the parental strands and synthesis of leading and lagging strands as in steps H-H. Although depicted as individual steps for clarity, unwinding and synthesis of leading and lagging strands occur concurrently.
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