Observation of DNA intertwining along authentic budding yeast chromosomes

Abstract

DNA replication of circular genomes generates physically interlinked or catenated sister DNAs. These are resolved through transient DNA fracture by type II topoisomerases to permit chromosome segregation during cell division. Topoisomerase II is similarly required for linear chromosome segregation, suggesting that linear chromosomes also remain intertwined following DNA replication. Indeed, chromosome resolution defects are a frequent cause of chromosome segregation failure and consequent aneuploidies. When and where intertwines arise and persist along linear chromosomes are not known, owing to the difficulty of demonstrating intertwining of linear DNAs. Here, we used excision of chromosomal regions as circular "loop outs" to convert sister chromatid intertwines into catenated circles. This revealed intertwining at replication termination and cohesin-binding sites, where intertwines are thought to arise and persist but not to a greater extent than elsewhere in the genome. Intertwining appears to spread evenly along chromosomes but is excluded from heterochromatin. We found that intertwines arise before replication termination, suggesting that replication forks rotate during replication elongation to dissipate torsion ahead of the forks. Our approach provides previously inaccessible insight into the topology of eukaryotic chromosomes and illuminates a process critical for successful chromosome segregation.[Keywords: DNA topology; chromosome segregation; sister chromatid intertwining; precatenanes; topoisomerase II; S. cerevisiae] Supplemental material is available for this article. Genetic information is encoded in DNA as a one-dimensional sequence of bases. However, it is the three-dimensional configuration of the DNA that directs how the information is accessed. This makes DNA topology a crucial player in genome function. The topological state of cellular DNA is under the control of DNA topoisomerases-enzymes that generate transient DNA breaks, rearrange, and then religate their substrates.

Plectoneme dynamics and statistics in braided polymers

“…Polymer braids lie at a crossroad between topology, statistical mechanics, and biological physics. Within cells, two DNA double-helices inevitably intertwine to form a braided molecule, or catenane, when replication terminates [1]. Braids also arise in biology with interwoven collagen helices [2], or amyloid fibrils [3].…”

Anaphase Bridges: Not All Natural Fibers Are Healthy

“…Early in vitro experiments showed that cohesin promoted catenation of DNA plasmids in the presence of Top2 [ 154 ]. The fact that the presence of cohesin bound to DNA prevents decatenation in vivo was first shown in yeast on DNA plasmids [ 60 , 61 ] and later confirmed also for endogenous chromosomes [ 55 ]. In higher eukaryotes, catenanes are specifically retained at centromeres, and they are resolved by Top2 only after that centromeric cohesin is cleaved prior to anaphase initiation [ 155 ].…”

“…Although fork rotation may be more frequent at specific regions, an elegant study in yeast by Uhlmann and colleagues showed that the resulting catenanes are free to diffuse after replication, as their occurrence was the same throughout the chromosomes. This group developed a system based on excision and circularization of chromosomal segments, which allowed to obtain the first direct evidence of catenation in endogenous linear chromosomes [ 55 ]. The only region that was found to be devoid of catenanes is the silent mating-type locus, maybe because its high level of compaction increases the stiffness of chromatin, thereby preventing the intertwines from diffusing in this region.…”

“…In agreement with the latter hypothesis is the finding that Top2 is enriched not only at the centromeres but also at other heterochromatic regions in mammalian cells [ 90 ]. However, this explanation is at odds with the finding that the yeast mating-type locus is devoid of catenanes [ 55 ] and the fact that a certain level of chromosome compaction is required for decatenation. Moreover, the enrichment of Top2 on heterochromatin may also be due to the role of this enzyme in promoting DNA compaction [ 57 , 88 , 91 , 92 ].…”

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

“…However, in budding yeast chromosomes, replication bubbles are bidirectional, and every bubble is serviced by a single replication factory, which precludes rotation of the individual replication forks. [ 73 ] Moreover, the Baxter's laboratory demonstrated that in S. cerevisiae , fork rotation and precatenation are restricted during DNA replication by Tof1/Csm3 and only occur in hard‐to‐replicate contexts. [ 12 ]…”