Kenneth J. Marians: Mechanisms of Replication Fork Reactivation

Orderly progression of the replication fork is essential for the timely and accurate duplication of our genetic material. Although the bacterial replisome is highly processive, capable of synthesizing megabase-long stretches of nascent duplex DNA without dissociating from the template, it is subject to many obstacles that can arrest replication forks. These obstacles can be chemical damage to the template bases (such as the formation of bulky adducts, abasic sites, or intra-strand thymidine dimers as a result of UV irradiation), nicks in one of the template strands, or frank double-strand breaks in the template. It is also the case that frozen protein-DNA complexes and trains of slow-moving RNA polymerases can block replication fork progression. In bacteria, the arrested replication forks must be restarted rapidly or asymmetric chromosome segregation will occur resulting in the production of chromosome-less, anucleate daughter cells. Repair of the DNA lesion may proceed replication restart or, as we have recently shown, the replisome may simply jump the damage and continue downstream. We have elaborated the biochemical pathways required for replication reactivation at several different DNA structures that can form when replication fork progression is arrested (Figure 1).

Stalled replication forks take two general forms, either the nascent leading strand is ahead of the nascent lagging strand or vice-versa (Figure 2). To study replication restart on stalled replication forks we devised a novel linear template that allows us to vary the structure of the forked end where the replisome will be loaded (Figure 3). Using this template, we have shown that there are two distinct biochemical pathways optimized for using these different substrates (Figure 4). These replication systems utilize the chromosomal replicase, the DNA polymerase III holoenzyme (Pol III HE), itself a 10-subunit enzyme, the single-stranded DNA-binding protein (SSB), and different combinations of the primosomal proteins that both recognize the stalled replication fork structures and re-load the replisome [PriA, PriB, PriC, DnaT, DnaC, DnaB (the replication fork DNA helicase), and DnaG (the primase)]. Our biochemical data demonstrated that the PriA system (composed of PriA, PriB, DnaT, DnaB, DnaC, and DnaG) prefers stalled replication fork structures where there is no gap in the nascent leading strand, whereas the PriC system (PriC, DnaB, DnaC, and DnaG) prefers stalled fork structures where there is a gap in the nascent leading strand. Because the PriA system also restarts replication at recombinant joint molecules that are generated after the formation of double-strand breaks in the template (Figure 2), these two replication restart systems are sufficient to restart any stalled replication fork in the cell. These biochemical observations agree with those of our colleague, Steve Sandler, who studies the genetics of replication restart.

Our studies on the biochemical pathways of replication restart suggested to us that DnaG could prime leading-strand synthesis de novo (see Figure 4 where, with the PriC system, even though a nascent leading strand is present at the fork, leading-strand synthesis appears to be dependent on the presence of the primase). This was a heretical concept. It had been accepted wisdom for over 40 years that leading-strand synthesis could only be primed at origins of replication. However, as shown in Fig. 5, both leading- and lagging-strand synthesis can be primed by DnaG de novo using the PriC system (and, for that matter, also the PriA system). This observation has led us to propose that replisomes that stall at a lesion in the leading-strand template can simply re-initiate replication downstream, leaving the template damage and a gap in the nascent leading strand behind (Figure 6). The damage can be repaired subsequently and the gap repaired by daughter-strand gap repair.

To be able to study these processes we have developed a new method for generating our linear template with a thymidine dimer in a specific location (Figure 7). Using these templates carrying DNA damage we can observe directly replication fork stalling and restart (Figure 8).

Among the questions that we are interested in answering are: What is the fate of the replication proteins when a replisome stalls? What is the structure of these stalled replication forks? What is the role of replication fork regression in restart? What are the requirements for replication restart? Do restart requirements differ depending on the type of DNA template damage? How do replication restart and lesion bypass cooperate? Is there a specific mechanism that displaces the Pol III HE from the DNA? And, can the same replisome jump template damage and restart downstream?