Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

Abstract

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.uring DNA replication, it is essential to completely unwind and remove all of the intertwining between the two strands of the template DNA double helix. This is achieved by the combined action of replicative helicases and topoisomerases. During elongation, replicative helicases force the strands apart, generating compensatory topological overwinding stress in the unreplicated region ahead of the fork. If overwinding accumulates, it prevents further DNA replication (1, 2). Relaxation of the stress is achieved either by topoisomerase action ahead of the fork, directly on the overwound region, or by coupling helicase action with rotation of the whole fork relative to the unreplicated DNA (Fig. S1). This latter pathway relaxes topological stress ahead of the fork at the expense of generating double-stranded intertwines behind the fork, often referred to as DNA precatenanes (3, 4). These intertwines are subsequently resolved by the action of type II topoisomerases. If type II topoisomerases do not completely resolve either the precatenanes or the full DNA catenanes formed at the completion of replication, the unresolved intertwines will cause chromosome bridging, nondisjunction, and aneuploidy (5). Fork rotation and DNA precatenation appear to be the primary pathway of unlinking when forks come together at the termination of DNA replication (6, 7). In addition, fork rotation appears to be a frequent event during elongation in vitro; it can support ongoing replication, and extensive precatenation is observed behind elongating forks (8-10). Therefore, the prevailing view is that the topological stress caused by DNA unwinding is reso...

The Causes and Consequences of Topological Stress during DNA Replication

“…However, evidence from eukaryotes indicates that fork rotation is actively limited outside of termination. Increasing the size of yeast plasmid replicons does not increase the average number of fork rotation events during DNA replication [22], arguing that fork rotation is not generally utilized outside of termination.…”

“…Deletion of either of the yeast homologues of Timeless/Tipin, Tof1/Csm3, dramatically increases the frequency of fork rotation during replication [22]. Therefore, these proteins are required to minimize fork rotation during DNA replication (in the context of Figure 1 Tof1/Csm3 promote usage of Figure 1A and inhibit Figure 1B).…”

“…The DNA bound complex could provide both a barrier to diffusion of topological stress and also occlude access of topoisomerases to the region between the replisome and the protein complex (Figure 2). Indeed, the addition of stable protein-DNA structures to episomal plasmids does increase the frequency of fork rotation during DNA replication [22]. Deletion of rrm3 and stabilization of protein-DNA binding, further increases fork rotation on stable protein-DNA plasmids [22].…”

“…Indeed, the addition of stable protein-DNA structures to episomal plasmids does increase the frequency of fork rotation during DNA replication [22]. Deletion of rrm3 and stabilization of protein-DNA binding, further increases fork rotation on stable protein-DNA plasmids [22]. This argues that the level of topological stress incurred at the replication fork on passage through stable protein-DNA is frequently sufficient to cause fork rotation (Figure 2).…”

“…However, our recent work has linked excessive fork rotation and DNA catenation with the incidence of DNA damage. We have shown that increasing the frequency of fork rotation and formation of DNA catenation by deleting tof1 appears to increase endogenous levels of DNA damage in cells and activates post-replication repair pathways [22]. DNA damage in this context is not caused by breakage of catenated DNA in mitosis since damage can be detected immediately following the S phase.…”