FACTORY vs. TRACK: THE MOVING REPLISOME DILEMMA

Unlike us eukaryotes, bacteria replicate their DNA differently. They have a circular genome that doesn’t form X-shaped chromosomes instead they have a pair of special machines called replisomes that start at a site called the origin of replication (ori), move bidirectionally through the arms of the genome, and end in the terminus (ter) opposite to the ori. This is a well-established fact, the question here was, How did the pair of replisomes move to replicate the DNA? Scientists came up with two theories, The “Factory” and the “Train” model. The Factory model said that the two replisomes are tethered together and anchored in one location acting as a stationary factory or moving together as a mobile factory. In contrast, the Track model suggested that the replisomes move independently along each of the arms and meet at the terminus.   

Factory vs. Track model of replication

To resolve this dilemma, scientists have attempted live-cell imaging to see if the replisomes actually split into two i.e. if they see two foci or just one, is it the track or the factory? In studies with E.Coli, B.subtilis, and Myxococcus xanthus, they have indeed observed two distinct foci. Then why is there still a dilemma? If there are two distinct foci then it definitely is the track model, right? Well, that is where a team of scientists from India and Switzerland pulled the brakes in their latest publication in Nature Communications: Chromosome Organization Shapes Replisome Dynamics in Caulobacter crescentus. They suggested that the results of these studies might be confounded because these bacteria can have more than a single round of replication (multi-fork replication) to reduce their replication times which means that the two distinct foci observed might just be two pairs of replisomes and not one separated pair. To address this dilemma, they used time-lapse imaging to measure the replisome dynamics and duplication of genomic loci in Caulobacter crescentus, a species of bacteria that is strictly restricted to only one round replication per cell cycle. Their findings suggest an elegant theory that it is neither just the factory nor the track model.  

Now how did they prove this? Getting into the details, when they imaged the replisome β-clamp subunit (DnaN) fused to superfolder GFP (sfGFP) in C.crescentus, they observed that most cells had one bright and one dim focus early during the replication (this is referred to as early splitting) while some had two bright foci at later stages of the replication (referred to as late splitting). To determine whether both the foci were functional replisomes, they fused sfGFP with single-stranded DNA binding (SSB) proteins which are an integral part of functional replisomes, and discovered that the dim DnaN focus did not have any SSB proteins near them. The same was repeated with other components of the replisome and the same was observed. Furthermore, they labeled sites in the left and right arms that are in proximity to the ori using orthogonal ParB/ParS systems to see if these regions moved away from each other to opposite cell halves before duplication suggesting a track model, although they observed that this did not happen and the sites moved only after duplication indicating colocalized replication, the factory model. When they imaged both DnaN and ParB (a protein essential for DNA segregation) using two-color imaging, they learned that the dimDnaN signals were residual replisome molecules that hadn’t yet fallen off during DNA segregation and appeared as a dim focus after segregation in the other half of the cell. They proved this by imaging DnaN and ParB in mutant cells that were only capable of partial DNA segregation. 

Now, that they established that the dimDnaN focus was not a functional replisome, what about the two bright foci in late-splitting cells? If it indeed was the factory model, how come there were two functional foci at the later stages of replication? Are the replisomes not directly linked together? They hypothesized that the replisomes could be indirectly linked together by chromosome interarm alignment. This interarm alignment is possible by SMC (Structural Maintenance of Chromosome) proteins that load onto the DNA near the ori. When imaging DnaN in SMC knockout strains of C.crescentus, they saw two bright foci during the early stages of replication along with SSB proteins indicating early splitting into two functional replisomes. This gave us a new theory that, although the replisomes started in a factory model, on disruption of inter-arm alignment the replisomes could decouple and switch to a track model. They concluded that even if there was a direct protein linker between the replisomes, the disruption of inter-arm alignment must be a stronger force that leads to the switching of models. 

There’s still a missing piece in the puzzle, in the SMC knockout strains, the replisomes split because of the disruption of chromosome inter-arm alignment, then why do the replisomes split in the later stages of replication in the wild-type cells, where and why are the inter-arm alignment affected in these cells? In the late-splitting cells, one replisome (in the left arm) seemed to move forward to the future division site faster than the other resulting in the splitting of the replisomes, this was seen by labeling 10 different sites of DNA in both the left and right arms (L1-L10, R1-R10). The parallel loci (R4 to R6) labeled on the right arm seemed to duplicate later than that of the left arm. At the end of replication, the foci seemed to colocalize again. On further inspection, they found that the genes present in these regions were highly transcribed, one of the genes present at these sites was the rsaA gene that translates into S-layer protein which makes up 31% of the proteome. Therefore they hypothesized that the replication-transcription conflicts in these loci resulted in slower progression of the right replisome resulting in disruption of inter-arm alignment and splitting of the replisomes. They tested this by adding a second rsaA gene in the opposite transcriptional orientation which resulted in the splitting of replisomes earlier than in the wild-type cells supporting their hypothesis.

The replisomes decoupling at the highly transcribed sites

Now that the final piece of the puzzle has been placed, we have a new theory that can be molded to fit any organism. It’s not solely the factory or the track model that efficiently completes DNA replication, but rather a dynamic combination of both. In C.crescentus the replication starts with a factory model with the replisomes being held together indirectly by the chromosome inter-arm alignment. The newly duplicated DNA is often immediately pulled to the other half of the cell in a process called DNA segregation with some DnaN still attached to it. However, this factory model switches to the track model when one of the replisomes encounters highly transcribed genes due to transcription-replication conflicts. The faster replisome advances to ter site and waits for the slower one to catch up. Replication comes to an end when both the replisomes colocalize again and disassemble. Therefore it's no longer Factory vs. Track, but Factory + Track. This provides us with a new perspective on replisome dynamics and how they are affected by chromosome organization.  

In the quiet choreography of a single cell, we glimpse the elegance of life itself.

REFERENCE:

Zhang C, Joseph AM, Casini L, et al. Chromosome organization shapes replisome dynamics in Caulobacter crescentus. Nat Commun. 2024;15:3460. doi:10.1038/s41467-024-47849-6. IMAGE CREDITS:

1. Cover Image: https://www.labmanager.com/dna-replication-under-the-microscope-27102
2. Factory vs. Track model of replication: https://www.researchgate.net/figure/Schematic-depiction-of-the-train-track-top-and-factory-bottom-models-for-Ecoli_fig1_335162731
3. The Replisomes decoupling at the highly transcribed sites: Reference