Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 25

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 relatively weak DNA base pairing in this AT-rich region allows the RNA transcript to form a transient but extended RNA–DNA hybrid (called R-loop) (Figure 2.9). This would displace a single strand of DNA that provides a prime target for loading of the helicase/primase complex, with subsequent loading and activation of both leading- and lagging-strand DNA polymerases (Figure 2.9). This would constitute a complete replisome like the ones discussed above, traveling away from the origin (in the rightward direction in Figure 2.9). Note that the lagging-strand DNA polymerase travels with the rightward replisome complex, as depicted by the dotted arrow in Figure 2.9 (and would actually be associated with that complex as discussed above). This initiation process has been recreated in vitro and requires the four T7 replication proteins along with the T7 RNA polymerase.

With the folding of the lagging strand around into a loop structure, one might expect that the replication-origin region would contain special features that create this loop. However, this is not the case. Very simple DNA substrates such as the one diagrammed in Figure 2.9 are converted into properly folded lagging-strand loops by the simple T7 replication machinery discussed above. Thus, the replisome complex discussed above has the intrinsic ability to create properly folded lagging-strand loops.

DNA replication from the T7 origin occurs in a “bidirectional” fashion, with one replisome generated in each of the two directions from the origin. Thus, after the first replisome is assembled and travels away from the origin as described above (the rightward direction in Figure 2.9), a second replisome complex is assembled for the leftward direction. In this case, the 3′ end of the first Okazaki fragment from the rightward direction is used as the primer for the new leading strand in the leftward direction (Figure 2.9). The precise pathway of protein loading for leftward direction is not yet clear, but the combination of a branch with a free 3′ end (from the first rightward Okazaki fragment) appears to be sufficient to trigger loading.