Algunas precisiones conceptuales y metodológicas

The human DNA polymerase ɛ complex is also a heterotetramer comprised of the PolE1 (also known as p261) catalytic subunit, p59 B

subunit and p12 and p17 accessory subunits. Conventionally, the catalytic N- terminus and structural C-terminus is conserved in human PolE1 too, with its B-subunit also binding to the zinc finger motifs present at its very periphery, while p12 and p17 heterodimer bind somewhat more centrally in the protein like in Xenopus (Tahirov et al., 2009). Unfortunately, because the less prescriptive nature of origins in humans makes the study of DNA initiation difficult, it is not clear whether PolE1 plays the same conserved role in origin firing. However, it has been shown to interact and be stimulated by the CMG component GINS, which could indicate a conservation of the Psf1-Dpb2 interaction that is so crucial in the essential origin firing activity of Pol ɛ in budding yeast (Bermudez et al., 2011). While crystal structures have been solved for PolE1-p59 interaction, it is still unknown what its N-terminal binding partner is (Baranovskiy et al., 2017). Interestingly, the EM structure of human GINS overlaps remarkably well with that seen in budding yeast, including the flexibility of the Psf1 subunit, indicating its interaction with another replisome component (Sun et al., 2015).

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PolE1 also has a strong resemblance to the budding yeast Pol ɛ complex, as it has been implicated in DNA repair (Moiseeva et al., 2016). Two separate, very early in vitro studies illustrated first that Pol ɛ co-purified

as part of a larger complex that mediated homologous recombination in response to double strand breaks, and then that it was proficient in

performing the gap-filling synthesis required after nucleotide excision repair alongside RPA, RFC, PCNA and DNA ligase (Jessberger et al., 1993, Shivji et al., 1995). Furthermore, the PolE1 C-terminus has also been found to interact with, and be stimulated in vitro by, Mdm2, the E3 ubiquitin ligase that targets the main tumour suppressor p53, and therefore being responsible for cell cycle regulation as well as DNA repair (Asahara et al., 2003). It is

hypothesised that Mdm2 could function to aid the transition of Pol ɛ reconfiguring from a replicative complex to one that repairs DNA, which could involve remodelling the protein composition at this site (Asahara et al., 2003). More recently, it was demonstrated that PolE1 undergoes

phosphorylation in its C-terminus in response to DNA damage, which was found to disrupt the binding of it to MMS19, a protein involved in Fe/S cluster assembly (Moiseeva et al., 2016). While the modification of PolE1 in

response to damage is no doubt interesting, it is very unclear what this phosphorylation could signify, as its abrogation does not entail greater sensitivities to DNA damaging agents, and neither is it understood what the relevance of an assembly of an iron sulphur complex at the polymerase could be (Moiseeva et al., 2016). Interestingly, while the C-terminus of PolE1 shares the same B subunit binding activities seen in other eukarya, its

expression is not able to suppress the deletion of the whole gene (Bermudez et al., 2011). This could indicate a greater need for the full catalytic activity of Pol ɛ in maintaining cell viability due to the size of the genome.

Owing to its essential nature as one of the major processive

polymerase, genetic disorders arising from mutations in PolE1 are extremely rare, although they do exist, as frequently, genetic disruptions simply would result in death. Two diseases have been linked to haploinsufficiencies of the PolE1 subunit, but each of these present with rather different phenotypes. One of these was discovered in 11 relatives, all of whom exhibited mild facial

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dysmorphism, immunodeficiency, livedo, and short stature (known as FILS syndrome) which was pinpointed to a homozygous single nucleotide

polymorphism in the intron of POLE1, which causes an alternative splice product that results in 90% of the resulting transcripts to be missing exon 34 (Pachlopnik Schmid et al., 2012). Interestingly, the sufferers of this

syndrome did not report higher levels of cancer susceptibility, but their

POLE1 insufficiency restricted the ability of numerous cell types to enter S phase and begin proliferation, which appeared to specifically affect the lymphocytes and osteoblasts, which would explain the immunodeficiency and problems relating to stature (Pachlopnik Schmid et al., 2012).

Interestingly, another patient outside of this initial family case study was also diagnosed with the exact same causative mutation, albeit with much more severe phenotypes (Thiffault et al., 2015). This variability in symptom

preservation was assumed to be the result of possible interactions between this haploinsufficiency with a pre-existing fault in the mismatch repair

pathway, thus creating a much more severe phenotype (Thiffault et al., 2015). Moreover, a parallel study in mice and humans illustrated that knockout of the Dpb4 ortholog in mice destabilised the entirety of the complex and this caused growth defects as well as defective B and T cell maturation, similar to those noted in FILS and related syndromes (Bellelli et al., 2018). Furthermore, at a cellular level, this loss of Pol ɛ caused defects in origin firing, replicative damage and genetic instability and remarkably, many of these were found to also be present in patient cells containing PolE1 mutations (Bellelli et al., 2018). This work simultaneously illustrates the heightened structural importance of the smaller accessory subunits in the mammalian Pol ɛ complex as well as hinting that the checkpoint and origin firing activities observed in budding yeast possibly being conserved through to humans.

Mutations in PolE1 have also been identified as germline mutations which can give rise to many cancer predispositions, including colorectal tumours (Palles et al., 2013). These mutations are localised to the exonuclease ‘proof-reading’ domain in the N-terminus of PolE1, which is consistent with the necessity of inherent genome instability that is crucial in

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the survival and evolution of cancer cells (Palles et al., 2013). More recently, mutations targeted in this exonuclease domain have also been characterised as an early feature of carcinogenesis in somatic endometrial and colorectal tumours (Temko et al., 2018). These mutations appeared before the tumours had become malignant, but their presence appeared to induce the genetic instability that then allowed subsequent driver mutations to arise, with many of these appearing to arise as a direct result of this impaired proof-reading mechanism (Temko et al., 2018). Furthermore, genetic screenings of PolE1 in colorectal cancer cell lines has identified that proof-reading mutations appear to emerge as a result an independent defect in mismatch repair, and together these produce high levels of genome instability for transformation (Yoshida et al., 2011). Another study sought to characterise several

exonuclease mutants, and assess how these mutations affected

tumourigenesis (Barbari et al., 2018). Interestingly, many of these mutants exhibited stronger mutator phenotypes than those observed in cells where the exonuclease domain has been removed completely, indicating that in order to drive the genome instability to push tumourigenesis, the function of PolE1 is affected in additional, unknown ways to this defective proof-reading (Barbari et al., 2018). Very few, if any mutations appear to have been

mapped to the C-terminus of PolE1, but having seen its conserved

importance throughout many eukaryotic organisms, this is unsurprising as disrupting this region would more than likely inhibit replication initiation in the cell and be lethal as a result.