Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 21

The inherent 5′ to 3′ directionality of DNA polymerase dictates that the leading- and lagging-strand products must be synthesized in opposite directions with respect to the parental double helix. Thus, for many years, models of the replication fork placed the lagging-strand polymerase quite far in space and uncoupled from the leading-strand polymerase (as in Figure 2.3). However, this is not an accurate view of the replication process.

A variety of experimental results instead show that the replisome functions as a unitary and well-coordinated multi-protein complex. Rather than being splayed apart from the leading strand, the lagging strand is looped around such that the two polymerases are bound together within the same protein complex (Figure 2.5). It is helpful to step through the cycle of lagging-strand synthesis to appreciate how this process works. As the synthesis of one Okazaki fragment is completed, the lagging-strand polymerase bumps into the 5′ end of the previously synthesized Okazaki fragment. This collision provides a signal that induces the DNA polymerase to dissociate from that segment of the lagging-strand. Note that the polymerase is still associated with the remainder of the replisome proteins upon dissociation of the completed Okazaki fragment. Evidence suggests that a second signal, perhaps acting as a backup mechanism, can also promote release of the lagging-strand segment by polymerase. This second signal is the synthesis of a new RNA primer for the next Okazaki fragment (Figure 2.5). Figure 2.5 does not differentiate whether the RNA primer is synthesized before or after DNA polymerase release, and likely either order is possible. In any case, the new RNA primer (primer 3 in the diagram), initially in contact with the helicase/primase, is now handed off to the DNA polymerase that just dissociated from the completed Okazaki fragment. Okazaki fragment extension by the DNA polymerase brings us back to where we started and completes the cycle (Figure 2.5). During replication of the T7 genome, which is roughly 40,000-base pairs long, this lagging-strand cycle occurs about 50 times on average. Because of the repeated cycle whereby the lagging-strand loop grows and shrinks, this mode of replication has been described as the “trombone model.”

Several features of the trombone model are worthy of note (Figure 2.5). First, during much of the cycle, two different regions of the lagging-strand template are single stranded — part of the loop (a segment that was just unwound by the helicase) and the segment between the active lagging-strand polymerase and the previous Okazaki fragment. Second, the loop starts off as a very small single-stranded segment and grows to include a large stretch each of single- and double-stranded DNA. The duplex DNA within the loop consists of the portion of the current Okazaki fragment that has been completed. Third, each of the single-stranded regions is coated with the ssDNA-binding protein (not shown in Figure 2.5), and so this protein must rapidly exchange and re-equilibrate on the strands as replication proceeds. Fourth, the lagging-strand polymerase remains associated with the replisome for multiple rounds of Okazaki fragment synthesis, rather than exchanging after each cycle as would be expected with the splayed-out replication fork in Figure 2.3. Experimental evidence for this conclusion includes the finding that an ongoing replication reaction continues many rounds of Okazaki fragment synthesis after being diluted extensively, to the point where any remaining free DNA polymerase in solution could not bind to the complex. Fifth, replication of the leading and lagging strands occurs at essentially the same rates, reflecting coordinated synthesis of the two strands.

Figure 2.5. The trombone model of DNA replication. The figure depicts one round of Okazaki fragment synthesis, with only the two DNA polymerases and replicative helicase shown for simplicity. Newly synthesized DNA is in red, and RNA primers (also red) are numbered. Note that the overall structures at the top and bottom are identical, except that one additional Okazaki fragment has been synthesized and the leading strand is extended accordingly. See text for detailed discussion of the steps in this model.

The looping model raises an important question — what happens if DNA polymerization on one of the strands is stalled or blocked? Does the polymerase on the other strand continue synthesizing DNA? This would create a dangerous situation, with a long stretch of ssDNA on the strand with the blocked polymerase and a complete uncoupling of the two DNA polymerases of the replisome. This question has been approached with a very special replication substrate using the T7 replication system (see “How did they test that?” at the end of this chapter). Polymerase extension on the lagging-strand template was blocked by incorporation of a special “chain-terminating” dideoxynucleotide only on that strand. Dramatically, synthesis of both the leading and lagging strands were strongly inhibited, demonstrating that the leading-strand polymerase halts when the lagging-strand polymerase is inhibited. Thus, the replisome is highly coordinated and the polymerases on the two strands somehow communicate with each other to maintain this coordination.

While a looped lagging strand is likely to be generally applicable to the process of DNA replication throughout biology, some of the Figures in the remainder of this book will show the “old-fashioned” splayed-out version for simplicity. Keep this in mind when you consider those simplified figures.