Replication Termination

Interestingly, while great strides have been made in delineating the step-wise assembly and gradual progression to origin firing, the

understanding of how replication terminates beyond two forks converging and the replisomes disassembling is comparatively less. A well-regulated termination mechanism is vital in maintaining genome stability through the prevention of re-replication. This process is much more difficult to study than other aspects of replication as it does not occur at a specific stage of the cell cycle, unlike origin firing and DNA synthesis, but instead as single events that are specific to the stretches of DNA being replicated and cannot be simply induced. In spite of this, various aspects of this process have been studied in eukaryotes, mainly through work performed in budding yeast, X. laevis egg extracts and Caenorhabditis elegans. This process was originally studied in Escherichia coli and in mammalian systems SV40 virus plasmid, which is replicated with eukaryotic factors except for encoding its own helicase (Sowd and Fanning, 2012). However, these do not provide good models for eukaryotic systems as their DNA is circular and their replication ends in a termination zone, which is genetically defined in E. coli but is positioned dynamically according to the site of the origin of replication in the SV40 system (Weaver et al., 1985).

In E. coli, this is dictated by termination zones made up of ten ter

sites, which each bind the Tus protein and stall the two forks after they have passed through the first five of these they encounter. Currently, the

mechanism of termination after the two forks encounter each other is

hypothesised to involve the two helicases passing each other after supercoil removal until they collide with the leading strand generated by the other replisome. At this point, it is believed that the helicase can then be unloaded by being substituted by a RecQ helicase, with the 3’ flap from the

encountered leading strand removed, resected and ligated together (Wendel et al., 2014). The mechanism of how the bacterial replisome is removed from

36

the DNA is still unknown, but phenotypes of Pol I mutants have implicated its role in the process (Markovitz, 2005).

In eukaryotic systems, this process is much more reminiscent of that observed in SV40 replication, as termination sites have been shown to be sequence nonspecific. In budding yeast, although early research focused on finding specific Ter sites, which provided moderate success in finding certain loci that possess fork pausing elements to assist fork convergence, the use of Okazaki fragment deep sequencing has identified many more sites that frequently occur midway between origins whose defining properties are the firing programme timings of the surrounding origins (McGuffee et al., 2013). This finding has also been confirmed in higher eukaryotes and is easily reconciled with our understanding of the less prescriptive nature of origin firing compared to prokaryotic systems therefore necessitating the nature of fork convergence to be more dynamic (Petryk et al., 2016). Due to this more stochastic nature of fork convergence in these organisms, much of the research focus has been on the mechanism of the replisome disassembly and removal that occurs. Once forks converge, it is believed that the oncoming helicases bypass each other, which could be permitted as their helicases are translocating on the two opposite leading strands, and

displacing each replisome onto the oncoming fork’s lagging strand (Fu et al., 2011). From here, it is unclear how the gaps between each replisome and the last Okazaki fragment of the lagging strand are then resolved by the polymerase machinery, as it is known that while this would take place on the de facto leading strand, Pol ε is unable to perform the strand displacement required for maturation, possibly meaning Pol δ could be reloaded to perform this sole function. Once these gaps are filled and ligated though, the

replisome is removed by a disassembly pathway newly discovered in

eukaryotes, with no known correspondence to prokaryotic or SV40 systems. Highly conserved fork disassembly pathways have been identified in both budding yeast and higher eukarya. Here, upon finishing replication, Mcm7 is polyubiquitinated (at lysine 27 in budding yeast, unknown in other organisms) by the SCF E3 ubiquitin ligase coupled with the specific substrate receptor Dia2 (SCFDia2_{) in budding yeast, and CRL2}Lrr1 _{in frog and worms}

37

(Moreno et al., 2014, Maric et al., 2014, Maric et al., 2017). This is then recognised by a segregase complex known as Cdc48 in yeast (p97 and CDC-48 in X. laevis and C. elegans, respectively), which disassembles the replisome from the DNA through the ATPase activity of its titular subunit (Maric et al., 2014, Moreno et al., 2014). Interestingly, C. elegans possesses a backup pathway of replisomal removal that occurs in the early stage of mitosis. Although its exact mechanism and whether it is present in other higher eukaryotes is not known. In this process, the CDC-48 segregase is assisted with a different co-factor, UBXN-3, and acts independently of CLR2LRR1 _{with regulation provided by a small ubiquitin-like modifier (SUMO)}

protease, possibly indicating a role for SUMO in place of the ubiquitinylation (Sonneville et al., 2017).

38

1.3 Checkpoint Signalling During Replication in Yeast