Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

Abstract

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

The many lives of type IA topoisomerases

“…In eukaryotes, replication termination has been proposed to be initiated by convergent fork rotation and stalling resulting from the accumulation of positive supercoils in the short un-replicated segment that can no longer be accessed by TOP1 or TOP2 (20,148). The ability of the dissolvasome to resolve late replication intermediates may be ideally suited to processing these structures formed during the latter stages of DNA replication (92,(149)(150)(151). Finally, it should be mentioned that the dissolvasome has been previously proposed to play a role in establishment of sister chromatid cohesion via a pathway that depends on the homologous recombination protein Rad51 and independently of other known cohesion pathways (152,153).…”

Supercoiling, R-Loops, Replication and the Functions of Bacterial Type 1A Topoisomerases

“…However, when replication is initiated at other sites on the chromosome, as is the case for R-loop-dependent replication, head-on conflicts may occur. Recent data support the involvement of yeast top2 and top3 in the resolution of topological problems related to conflicts between replication and rDNA transcription [99]. In E. coli, as it is the case for replication fork convergence, topo IV and topo III might be involved in the resolution of such topological problems.…”

Common Features of the Pericentromere and Nucleolus

“…In budding yeast, TERs are particularly concentrated near centromeres, and mutation of Top2 causes double-stranded breaks and recombination at these sites [138]. Top2 mutants result in repair checkpoint activation that is counteracted by a Top2-Fob1 double mutant, implying that Top2 mediates proper replication termination in rDNA as well [139].…”