Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies. Kenneth N Kreuzer
a very similar replisome structure forms with three copies of full-length helicase/primase, three copies of truncated helicase/primase, and three copies of DNA polymerase.
Considering the positions of the leading- and lagging-strand polymerases, the third (unengaged) DNA polymerase (bottom right in Figure 2.6B) is in prime position to take over synthesis on the lagging strand should the lagging-strand polymerase become disabled or dissociate. It will be interesting to deduce the structural transitions that occur during this proposed polymerase-switching process.
With these remarkable advances in the T7 replication system, we are now in a position where we can contemplate how the trombone model plays out in the context of an actual three-dimensional structure of a protein complex of some 600,000 Daltons. Using the structural model (Figure 2.6B) and cartoon (Figure 2.6C) as guides, try to visualize how the parental, leading, and lagging strands slide through the complex during the various steps of DNA replication.5
2.7T7 ssDNA-binding protein helps to organize the replisome
The transient but fairly extensive regions of ssDNA on the lagging-strand template are blanketed by the ssDNA-binding protein, which both protects and organizes the DNA. The dynamics of protein binding to these single-stranded regions is fascinating. As mentioned above, ssDNA-binding protein has an acidic C-terminal extension. Indeed, 15 out of 26 residues in this extension are aspartic or glutamic acid. When free in solution, the ssDNA-binding protein forms a dimer in which the acidic C-terminal extension of each monomer binds to the (basic) DNA-binding site within the body of the opposite subunit (Figure 2.7). Binding of the C-terminal extension to the DNA-binding site presumably shields the protein from binding other negatively charged molecules in the cell and modulates the affinity of the protein for ssDNA.
Figure 2.7.Protein–protein interactions centered on the T7 ssDNA-binding protein. The acidic C-terminal tail of the protein is critical for interactions with itself and other proteins. In solution, the protein dimerizes via mutual interactions between the tail of one protein and the body of its partner. Once bound to ssDNA, the acidic C-terminal tail becomes available for interactions with the DNA polymerase and with the helicase/primase, as shown at the bottom of the figure.
The C-terminal extension of the ssDNA-binding protein is the region that binds the T7 DNA polymerase and helicase/primase. This implies that the dimer in solution has weakened interactions with these replication proteins. However, upon binding to ssDNA, the C-terminal extensions are displaced from the DNA binding sites and thereby become available for interactions with the other replication proteins (Figure 2.7). Overall, this simple but elegant architecture of the ssDNA-binding protein allows the protein to be readily available in solution to bind any ssDNA that is generated, and yet not sequester the much more limited amounts of replication proteins. Also, the architecture presumably ensures that free protein does not bind extensively to the polymerase and helicase/primase at the replication fork, allowing preferential binding of ssDNA-binding proteins that are bound to the ssDNA within the fork.
Through its interactions with the DNA polymerase and helicase/primase, the ssDNA-binding protein plays a central role in coordinating and regulating the functioning of the replication machine. The protein modulates the activities of both of these proteins and is critical for efficient lagging-strand synthesis. Indeed, when the ssDNA-binding protein is withheld from in vitro replication reactions, lagging-strand synthesis is strongly inhibited, with abnormally short Okazaki fragments, and coordination between leading- and lagging-strand synthesis is lost.
2.8Finalizing the lagging-strand product
The reactions described above can provide a fully intact, duplex DNA molecule as the leading-strand product. However, the lagging-strand product will consist of one intact parental strand along with a product strand that has an interruption and a small segment of RNA where each Okazaki fragment was initiated. These imperfections are repaired following the main replication reaction to generate the fully intact DNA products (Figure 2.8).
Figure 2.8.Okazaki fragment processing. The sequential action of a 5′ to 3′ exonuclease, DNA polymerase, and DNA ligase result in removal of the RNA primer and sealing of adjacent Okazaki fragments into an intact strand.
An additional T7 protein6 is a specialized nuclease with two key activities. First, this protein is a 5′ to 3′ exonuclease and plays an important role in removing the RNA primers at the 5′ ends of each Okazaki fragment. Second, the protein has an activity called “flap endonuclease,” which can cleave off a short segment of extra DNA or RNA at a nick. We will return to a discussion of flap endonucleases when we discuss eukaryotic DNA replication in Chapter 4.
In addition to processing Okazaki fragment ends, the T7 exonuclease also plays a key role in degrading the bacterial host DNA during T7 infection. T7-encoded nucleases degrade the host DNA in order to re-utilize the resulting nucleotide residues for synthesizing its own DNA. Since the T7 genome is less than 1% the length of the bacterial (E. coli) genome, this is an important source of precursors that can fuel the synthesis of many phage genomes.
The action of the T7 exonuclease on the 5′ ends of each Okazaki fragment temporarily leaves a short gap of ssDNA, which is filled in by DNA polymerase (Figure 2.8). Once the gap is bridged so that the product strand has two adjacent nucleotide residues, DNA ligase seals the nick and the lagging strand is complete. T7 encodes its own DNA ligase,7 but T7 mutants lacking that enzyme can instead use the host DNA ligase for Okazaki fragment maturation.
2.9Back to the beginning — How does T7 DNA replication initiate?
The elegant dynamics of the replication process discussed above begs the question of how the process is initiated. T7 DNA replication initiates at a defined location in the bacteriophage genome, where the replication machinery must be loaded. The so-called replication origin consists of two tandem promoters for T7 RNA polymerase-induced transcription and an AT-rich region of DNA just downstream (Figure 2.9A). Importantly, the RNA polymerase transcripts are used as the primer for leading-strand synthesis during in vitro reactions and presumably also in vivo.
Figure 2.9.Model for initiation of bacteriophage T7 DNA synthesis. RNA polymerase generates a transcript from either of two transcriptional promoters in the origin region. In this model, the RNA transcript forms a stable RNA–DNA hybrid, called an R-loop. The displaced strand of DNA is used as a loading site for the helicase/primase, and the RNA transcript is used as primer for DNA synthesis in the rightward direction. The DNA polymerase that completes the first Okazaki fragment is associated with the rightward helicase complex (indicated by dotted arrow) and travels with that complex in the rightward direction (this diagram does not present the folded structure of the trombone model for simplicity sake). After rightwards replication has exited the origin region, a new leftward replisome is assembled to complete the establishment of bidirectional DNA synthesis. New molecules of helicase and DNA polymerase assemble on the branched DNA at that site, with the 3′ end of the first (rightward) Okazaki fragment serving as primer for the leftward leading strand. In this figure, RNA residues are in green and newly synthesized DNA is in red.
Why is the AT-rich region just downstream of the promoters necessary for origin function? One model is that the