MCB 201 Gene Expression - Spring Semester 2006
Lecture 9 (DNA replication cont.)
6. Figure 4-34. Panel A. Model of an SV40 DNA replication fork and assembled proteins: in vitro replication of SV40 viral DNA . Replication of eukaryotic DNA viruses has served as a model for studying eukaryotic DNA replication. This is because DNA viruses have a well-defined origin of replication and generally use the same proteins that cells use to replicate their DNA. Below is a step-by-step outline of replication of the SV-40 eukaryotic DNA virus:
(1). The viral protein called T-antigen (Tag) binds to the origin and melts the DNA. T-antigen hexamer is a helicase. Unwinding of the double helix of DNA by Tag establishes a replication fork and sets a direction of fork movement. The polymerase delta that synthesizes the leading strand moves in the same direction as the large T-antigen complex (helicase).
(2). Replication protein A (RPA) is a single strand DNA binding protein that stabilizes ssDNA after melting of the double helix.
(3). Eukaryotic primase binds to the ssDNA together with DNA polymerase-alpha. The eukaryotic primase makes a short RNA primer which is then extended by DNA polymerase-alpha. RFC, replication factor C binds to and stimulates Polymerase-alpha activity. Thus, lagging strand synthesis is started discontinuously by a complex of polymerase-alpha, primase and RFC.
(4). The protein proliferating cell nuclear antigen (PCNA) replaces DNA polymerase-alpha with the highly processive DNA polymerase-delta. Remember that processive means that this polymerase can copy long stretches of template without falling off. Not surprisingly, polymerase-delta also synthesizes the leading strand. Not surprisingly, polymerase delta also synthesizes the leading strand.
Panel B: The three subunits of PCNA form a collar with a central hole through which double-stranded DNA passes. The ribbon model of the PCNA protein traces the peptide backbone of these subunits.
Panel C: Two views of the large subunit of RPA. On the left, note how the two binding domains of RPA extend the single strand of DNA, exposing the bases in an optimal configuration for binding by a DNA polymerase. The view on the right shows how RPA wraps around the DNA strand.
7. Figure 4-35. Electron microscopy of replicating SV40 DNA indicates bidirectional growth of DNA strands from an origin. Demonstration of bidirectional chain growth from a single origin in viral DNA. At increasing times, samples of infected cells are taken, the circular viral DNA is extracted and cut with a restriction enzyme (EcoRI) that makes only one specific cut in the viral DNA. The results are then viewed by electron microscopy. The distance from the ends of the cut DNA to the center of the replication 'bubble' was found to be constant at all times even though the bubble grew in size around this center as replication progressed, indicating that replication is bidirectional from a common origin located at the center of the bubble.
Media Connection: Bidirectional DNA synthesis
8. Figure 4-36. Bidirectional mechanism of DNA replication. This is a simpler diagram based on Figure 4-34, with the left replication fork in this diagram comparable to the fork in figure 4-34. Here the action of the large T-antigen helicases and the coordination of the leading and lagging strand synthesis is emphasized. Note that this is diagram is drawn for clarity and does not reflect the fact that unwinding and synthesis occur at the same time. How do you find the sites of leading and lagging strand synthesis in this diagram? The first primers synthesized 'prime' or allow the start of leading strand synthesis by extension of their 3' ends. Now look at the replication origins to find the sites where primer is laid down to start the lagging strand. Each time the replication fork moves, a new piece of template DNA is revealed for RNA primer synthesis, and this becomes another start site for lagging strand DNA synthesis.
9. Figure 12-5, Lodish 4e: Consensus sequence of the minimal bacterial replication origin based on analysis of genomes from six bacterial species. We can define a replication origin as a segment of DNA in the genome that is necessary and sufficient for replication of a DNA molecule. The E. coli replication origin has been intensively studied. It consists of about 240 base pairs of DNA at the start site for replication. E. coli oriC and a number of other bacterial origins of replication contain two types of repetitive sequences, a 9-bp sequence (9-mer, DnaA protein binding site) and an AT-rich 13-bp sequence (13-mer, easily dissociated DNA double helix). The high AT content facilitates local melting of the double helix to allow the DNA replication complex to assemble on the single stranded DNA.
10. Common properties of origins of replication.
We can generalize to identify three common features of replication origins:
A. Unique DNA segments that contain multiple short, repeated sequences.
B. Short repeat units are recognized by multimeric (more than one protein subunit) origin-binding proteins. These proteins control the initiation of DNA replication by directing assembly of the replication machinery to specific sites on the chromosome.
C. Usually contain an AT-rich stretch to facilitate unwinding of double stranded DNA.
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