The Causes and Consequences of Topological Stress during DNA Replication

Abstract

The faithful replication of sister chromatids is essential for genomic integrity in every cell division. The replication machinery must overcome numerous difficulties in every round of replication, including DNA topological stress. Topological stress arises due to the double-stranded helical nature of DNA. When the strands are pulled apart for replication to occur, the intertwining of the double helix must also be resolved or topological stress will arise. This intrinsic problem is exacerbated by specific chromosomal contexts encountered during DNA replication. The convergence of two replicons during termination, the presence of stable protein-DNA complexes and active transcription can all lead to topological stresses being imposed upon DNA replication. Here we describe how replication forks respond to topological stress by replication fork rotation and fork reversal. We also discuss the genomic contexts where topological stress is likely to occur in eukaryotes, focusing on the contribution of transcription. Finally, we describe how topological stress, and the ways forks respond to it, may contribute to genomic instability in cells.

Keywords: DNA replication; DNA topoisomerases; DNA topology; fork reversal; fork rotation; transcription.

Figure 1

Resolving the topological stress generated by replicative helicase action. During elongation of DNA replication, unwinding of the parental template by the replicative helicase (CMG) separates the parental strands, but does not resolve the intertwining that exists between them. The intertwines between the strands are displaced into the region ahead of the fork leading to overwinding and positive (+) helical stress (shown in figure as positive supercoils). There are two pathways to relax this stress before it arrests ongoing DNA replication—topoisomerase action and fork rotation. (A) The tension is directly resolved by the action of either a type IB topoisomerase (such as eukaryotic topoisomerase I (Top1)) or a type II topoisomerase (such as eukaryotic topoisomerase II (Top2)). These topoisomerases act effectively as “swivelases” ahead of the fork. (B) Champoux and Been (see text) proposed a second mode of unwinding where the helical tension is relaxed by rotation of the fork to generate catenated DNA sister-chromatid intertwines behind the fork. Although these intertwines should not arrest forward elongation of replication, it is essential that the decatenating type II topoisomerases resolve all DNA catenation before the completion of cell division. (C) At the termination of DNA replication when two forks converge, topoisomerases become sterically excluded from the unreplicated DNA. In this case the final few turns of DNA have to be unwound by rotation of the fork relative to the DNA. In eukaryotes the CMG helicase remains bound until they reach the replicated DNA on the other side of the termination zone. Therefore, the two CMG helicase complexes and the leading strand polymerase bypass one another during termination.

Figure 2

Hypothetical model of induction of topological stress at a stable protein-DNA complex. As the replisome approaches a stable protein-DNA complex, topoisomerases are inhibited from acting between the complex and the converging replisome. Potentially, this will initiate fork rotation to facilitate unwinding. In this case replication fork elongation will also be facilitated by the action of an accessory helicase, such as Rrm3, which will promote displacement of the stable protein-DNA complex.

Figure 3

Hypothetical models of the consequences of topological stress and head-on replication-transcription conflicts in eukaryotes and E. coli. Head-on collisions between the DNA replication machinery and active transcription will lead to high levels of positive (+) super-helical stress ahead of the fork. There will also be negative (−) superhelical stress behind the transcription bubble. Analogous to the termination of DNA replication, the converging replication and transcription machineries will progressively sterically occlude topoisomerases from acting on the positive (+) super-helical stress between the complexes, inhibiting unwinding. This is likely to initiate replication fork rotation. As the replication and transcription machineries fully converge there are likely to be distinct consequences in eukaryotes (A) compared to E. coli (B). (A) In eukaryotes both the CMG helicase and the RNA polymerase translocates 3′-5′. These will converge on different strands. If the complexes act the same way as converging complexes in termination they will bypass one another. (B) In contrast, in E. coli the replication helicase DnaB translocates 5’-3’, travelling on the same strand as the converging RNA polymerase travelling 3′-5′. Therefore, these complexes will collide unless the RNA polymerase is displaced.

Figure 4

The hypothetical model of fork reversal in eukaryotes in response to DNA topological stress. Replication forks have been observed to reverse in response to high levels of topological stress (e.g., induced by camptothecin (CPT)). In this case the paused replisome must; (i) partially dissemble to expose the nascent strand, (ii) displace the nascent strands (potentially actively); and (iii) promote parental strand re-annealing. The reforming of the intertwined parental strand will relax the positive helical stress ahead of the fork by rotation of the strands and bound CMG helicase. Potentially, this is a stable structure, which will require active structuring to re-form an active fork structure.