Y-family DNA polymerases and their role in tolerance of cellular DNA damage

  Julian E Sale1, Alan R Lehmann2 and Roger Woodgate3

A finely tuned and complex molecular machine replicates DNA with very high efficiency and astonishing fidelity. However, the price paid for this is that it is easily disturbed by damage on the DNA template. Despite the plethora of mechanisms to repair DNA, it is likely that the replication machinery will encounter lesions in the DNA template during each cell cycle. The catalytic site of the replicative DNA polymerases is compact and intolerant of most DNA lesions. As a consequence DNA synthesis arrests at most forms of DNA damage. This poses a considerable problem for the cell - it must replicate the damaged DNA before mitosis so that a complete copy of the genome is passed to both daughter cells. The solution adopted by cells in every branch of life is known as DNA damage tolerance: DNA is synthesised past the damaged bases, which can be subsequently excised once safely located within duplex DNA, after the replication fork has passed. Direct replication past DNA damage in these circumstances, a process known as translesion DNA synthesis (TLS), is carried out by specialised DNA polymerases, the most abundant class of which are those belonging to the Y-family¹. As detailed in Table 1 there are two members of this family in Escherichia coli and Saccharomyces cerevisiae and four members in mammalian cells.

Drawing on examples across all domains of life, this review will examine the principles that govern the ability of the Y-family polymerases to synthesise past damaged DNA and yet allow the cell to restrict the potentially damaging mutagenic activity of these enzymes. After a brief historical introduction, we  focus on the underlying biochemical and structural features of the family and then examine the mechanisms that control their activity. Finally, we summarise the roles of the Y-family polymerases in a number of other processes to illustrate how their properties have been co-opted to meet specialised needs within a cell.

A brief history

Special mechanisms required to replicate past DNA damage were first identified by Rupp and Howard-Flanders in the late 1960s, who showed that, in E. coli, gaps were formed in newly synthesised DNA after UV-irradiation and subsequently sealed². Although most of these gaps were filled-in by a recombination-mediated “damage avoidance” mechanism, the idea that some of them were filled by an error-prone DNA polymerase was mooted in the early 1970s³. The REV (reversionless) loci in budding yeast⁴ and umu (UV nonmutable) loci in E. coli^{5, 6} were postulated to be involved in this error-prone bypass pathway and they were thought (erroneously as it turned out), to somehow lower the fidelity of the replicative polymerases (Supplementary Information S1 (Box)) to allow them to replicate past damaged bases. At about the same time the variant form of the sunlight-sensitive cancer-prone genetic disorder xeroderma pigmentosum (XPV) was shown to be caused by a reduced ability to make intact daughter DNA strands following UV-irradiation⁷.  

It was more than two decades later that the products of these and related genes were identified. In yeast, the product of the REV1 and REV3 genes were found respectively to be a dCMP transferase⁸ and DNA polymerase ζ, a B-family polymerase, which was capable of bypassing the major UV photoproduct, the cis-syn cyclobutane thymine dimer, with about 10% efficiency⁹. Rad30, was subsequently also shown to be a bona fide DNA polymerase, termed polh, that could bypass cis-syn cyclobutane thymine dimers, very efficiently and relatively accurately¹⁰.  By the end of 1999, a human homolog of Rad30 had been identified and was shown to be the product of the gene defective in XPV cells ^{11, 12}. At roughly the same time, both the E. coli DinB protein and UmuD'₂C complex were also shown to be bona fide TLS polymerases, called E.coli polIV¹³ and polV^{14, 15} respectively. Shortly thereafter, a second human ortholog of yeast Rad30 and a human ortholog of E.coli DinB were identified and shown to encode novel TLS polymerase poli^16-19 and polk^20-23 respectively. Thus, within a period of about 18 months, the field underwent a seismic change from having no clear understanding of the mechanisms of TLS to a defined enzymatic process that is facilitated by specialized DNA polymerases conserved from bacteria to humans. The basic steps of TLS, as shown in Figure 1 and described in more detail below, involve several “polymerase switches”. First, the stalled replicative polymerase must be displaced and replaced with a TLS polymerase, which inserts either correct or incorrect bases opposite the lesion. Extension from the (mis)incorporated base may be performed by the same polymerase, or a second TLS polymerase. Finally, when base-pairing has been restored beyond the lesion, the replicative polymerase regains control^{18, 24, 25}.

The presence of these low-fidelity polymerases in all domains of life, and indeed their expansion in higher organisms, suggests an essential evolutionarily conserved role²⁶. All cells are exposed to DNA insults from both endogenous and exogenous sources, and the Y-family DNA polymerases allow them to tolerate potentially lethal damage.  Sometimes, but not always, however, tolerance is accompanied by unwanted mutagenesis, which can have deleterious consequences. It is crucial therefore to strictly regulate the activity of the Y-family polymerases, so that they are not deployed inappropriately.

Structural and biochemical features

The key differences between Y-family polymerases (Table 1) and most other polymerases are their ability to replicate past damaged bases and their reduced fidelity on undamaged templates. The features of the polymerases that confer these properties are described in this section.

DNA polymerase fidelity

The structural features of the replicative polymerases are geared towards maximising replication efficiency and fidelity (Supplementary Information S1 (Box)). Surprisingly polymerase fidelity, defined as the ratio of incorporation of the correct over incorrect nucleotide, is determined largely by the efficiency of incorporation of the correct nucleotide rather than efficiency of incorrect nucleotide insertion, which varies relatively little between polymerases²⁷. The general structure of the replicative DNA polymerases has been likened to a right hand, with palm, fingers and thumb domains (Figure 2A).²⁸: The catalytic carboxylate-metal ion complex lies within the palm domain, while the mobile fingers and thumb domains grasp the template and primer to create a tight active site intolerant of misalignment between the template base and incoming nucleotide^{29, 30} (Fig. 2A). The fingers domain undergoes a significant conformational change during the catalytic cycle as explained in Supplementary Information S1 (Box).

General features of the catalytic domains

Despite poor sequence conservation with the replicative polymerases, the structures of Y-family polymerase catalytic domains reveal a similar overall ‘right hand’ topology. However, their active site is more capacious than that of the replicative polymerases, allowing the accommodation of a bulky adduct on the template base. The finger and thumb domains are stubbier, making fewer DNA contacts with both the DNA and incoming nucleotide, which contributes to the enzymes’ decreased processivity and poorer fidelity. The Y-family polymerases have an additional domain, termed the “Little Finger”, which also mediates DNA contacts close to the lesion site. Indeed, this domain has been implicated in polymerase selectivity for certain lesions³¹. While these features allow nucleotide incorporation opposite damaged bases, they also militate against accurate and processive replication, and the mutagenicity of the Y-family polymerases is further compounded by a lack of the 3’-5’ proofreading exonuclease activity characteristic of replicative polymerases (Compare Figs 2B and C with Fig 2A).

Y-family acrobatics

Within these general principles, the Y-family polymerases adopt an impressive array of novel mechanisms to replicate over a diverse range of DNA lesions, an ability that extends even to bypassing short stretches of non-DNA carbon chain ³². Next, we illustrate some of these approaches and features by examining how the structural solutions adopted by Y-family members explain their biochemical properties and ability to replicate particular lesions (Table 1). As mentioned previously and shown in Figure 1, TLS is a multi-step process involving (mis)incorporation opposite the DNA lesion and subsequent extension past the lesion (Fig.1). The polymerases thought to be involved in these different steps in E. coli, S. cerevisiae and human cells are detailed in Figure 3. The eukaryotic B-family enzyme Polz is especially suited to carry out the extension past the DNA lesion.

Dpo4: a model for PolIV/DinB/polk-like polymerases

Solfolobus solfataricus Dpo4 (Fig. 4) was the first Y-family DNA polymerase to be crystallised in a ternary complex with DNA and incoming nucleoside triphosphate³³. In the intervening ten years, Dpo4 has been crystallised in the process of facilitating TLS past a broad spectrum of DNA lesions. These structures indicate that Dpo4 is very flexible and can accommodate a plethora of lesions in its active site through a variety of contortions of the primer, template, or incoming nucleoside triphosphate. Interestingly, however, the only lesion that Dpo4 bypasses very efficiently is an abasic site.  Efficient bypass is achieved by displacement of the abasic site into an extra-helical position, with the 5’ undamaged base serving as the template for synthesis³⁴. This results in a -1 frameshift mutation if the primer template does not realign after TLS and provides the structural basis for the high frequency of –1 bp mutations generated by Dpo4 and orthologs, such as E.coli pol IV³⁵ and human polk³⁶.

Dpo4 also uses a similar mechanism of looping out the much larger benzo[a]pyrene diol-epoxide (BPDE) adduct on dexoyguanosine³⁷. The bulky lesion is flipped into a structural gap between the little finger domain and the core domains, so that the correct geometry for TLS occurs.  Archaeal Dpo4 and human polk have strikingly similar structures despite rather poor sequence conservation, with all domains superimposable³⁰. However, subtle differences exist.  In particular, the gap between the little finger domain and the catalytic core is enlarged, which helps explain why these enzymes are able to bypass a BPDE lesion in vitro²³ and in vivo³⁸.

Molecular splinting by Polη

Polη is particularly efficient at replicating cyclobutane pyrimidine dimers (CPD). It can do so alone and with high efficiency, incorporating A-A opposite a cis-syn T-T CPD with similar accuracy to unmodified T-T¹⁰. This provides a ready explanation for the features of polh-defective XP-V cells and patients. In the absence of polh, other polymerases presumably substitute but with lower accuracy, resulting in higher UV-induced mutagenesis and carcinogenesis. Evidence suggests that either poli or polk together with polz are likely candidates ^{39, 40}. A recent set of structures catching yeast and human polη in the act of bypassing a CPD reveal a number of features that enable this polymerase to be so effective at replicating this common lesion^{41, 42}. The active site of polη is particularly large and is able to accommodate both of the linked thymine bases of the template (Fig. 2C). The CPD is further stabilised such that the linked Ts can pair with the correct incoming dA, thereby allowing the polymerase to extract the correct coding information from the lesion. Since the CPD remains in the duplex after replication, it will continue to introduce distortion, which could contribute to slippage and frameshifts as the newly replicated duplex emerges from the catalytic site. Polη counters this by providing a continuous positively charged molecular surface, created by a specialised β-strand in the little finger domain, which acts to splint the newly synthesised duplex into a stable B-form. Thus, the enzyme is not only able to accurately synthesise across T-T CPDs, it also ensures the reading frame is maintained. Finally, when the CPD emerges from the active site, and the cell is in danger of replicating undamaged DNA with low fidelity, steric clashes ensure that the DNA is displaced from the enzyme, when three bases have been inserted beyond the lesion⁴².

Poli’s structure confers its unique mutagenic signature

Human poli is unique amongst Y-family polymerases in exhibiting a 10⁵-fold difference in fidelity depending upon the template base. When copying dA, the enzyme efficiently incorporates the correct base dT, with a respectable misincorporation fidelity of 1-2 x10^-4. However, when replicating dT, the enzyme misinserts dG 3-10 times more frequently than the correct dA.  How can an enzyme be reasonably accurate when copying dA, but highly error-prone when copying dT? The answer lies in the unique active site of poli and in particular, key residues in the finger domain that restrict positioning of the templating base.  Structural studies reveal that template A is driven into a syn (rather than the normal anti) conformation by Gln 59 and Lys 60 from the finger domain. In such a conformation, there are few hydrogen-bonding opportunities with any incoming nucleotide other than dT in an anti, or so-called “Hoogsteen” conformation⁴³. The same finger domain residues that are important for accurate replication of template dA, are responsible for the high misincorporation seen at template dT⁴⁴. Side chains protrude into the active site of poli and restrict its size.  As a consequence, the template dT is always held in the anti conformation irrespective of the incoming nucleoside triphosphate. While incoming dA is in the syn conformation and exhibits reduced base stacking, misincorporated dG is in an anti conformation and the mispair is further stabilized by hydrogen bonds on Gln59⁴⁴.  This restrictive feature of the active site facilitates the correct replication of the important oxidative lesion 8-oxoguanine⁴⁵. 8-oxoguanine can adopt two alternate conformations (anti or syn) and as a consequence, with most polymerases, it can pair equally well with either dC or dA. However, poli restricts it to the syn conformation, preventing the dual coding properties of the lesion by inhibiting the syn–anti [OK? Yes The journal style does not allow the use of slashes.] conformational equilibrium and promoting formation of the most stable and correct base pair with dC⁴⁶. These properties may explain the participation of poli in a specialized TLS pathway within the mechanism of base excision repair ⁴⁷.

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Base flipping during deoxycytidyl transfer by REV1

The catalytic activity of REV1 is, unusually, restricted to the insertion of dC opposite template dG^{8, 48} or a limited range of lesions including abasic sites and bulky N²-dG adducts^{49, 50}.  While loss of the catalytic activity of REV1 has no discernable defect on survival of DT40 cells following DNA damage⁵¹ or murine development⁵², it has recently been shown to be required for the ability of budding yeast to survive exposure to 4-nitroquinoline-1-oxide (4-NQO)⁵³. Furthermore the mutation spectrum at abasic sites is altered in mutants lacking the catalytic activity of REV1 generated either during immunoglobulin gene somatic hypermutation in vertebrate cells^{52, 54, 55} or in yeast cells during abasic site bypass⁵⁶ . This confirms an in vivo role for the dCMP transferase activity of REV1. The crystal structure⁵⁷ revealed that, while the enzyme is able to detect the presence of a template dG and select for an incoming dC, it does not do this by detecting correct base pairing. Instead, the template dG is swung out of the helix and temporarily coordinated by a specialised loop within the Little Finger domain. The space previously occupied by the template G, or provided by an abasic site, is instead filled by Arg324 of REV1, which forms hydrogen bonds with the incoming dCTP. This mechanism therefore allows the bypass of bulky dG adducts whilst retaining the specificity for incorporation of the correct dC base.

Regulation of the Y-family polymerases

While the biochemical activities of the Y-family polymerases promote cell survival by allowing the release of replication blocks, they are also potentially deleterious to the cell. Thus, their access to sites of DNA synthesis must be carefully regulated to avoid unscheduled or extensive mutagenic polymerisation. Managing the balance of appropriate recruitment to undesirable activity is a problem facing all organisms, and despite the apparently different approaches taken to regulate translesion polymerases in prokaryotes and eukaryotes, common themes emerge. In all organisms there appears to exist a spectrum of regulatory mechanisms defined at one end by a simple ‘mass action’ approach, in which the number of active polymerase molecules is regulated, through to exquisitely specific control of protein-protein and protein-DNA interactions. In this section we will explore the strategies for regulating the Y-family polymerases from prokaryotes to mammals.

Regulation of cellular concentrations

The conceptually simplest method for regulating the action of the Y-family polymerases is to adjust their cellular concentrations (Figure 5A). This approach is central to the SOS response in E. coli, during which ~40 genes are up-regulated by DNA damage⁵⁸. Among these genes are those encoding the two Y-family polymerases, DinB (polIV) and UmuDC (polV) (Figure 4)⁵⁸. However, transcriptional regulation alone is just one level that E. coli utilizes to keep the polymerases under control. Error-prone polV, made up of heterotrimeric UmuD’₂C, is subject to multiple further levels of regulation. First, both UmuD and UmuC proteins are rapidly degraded by the Lon protease⁵⁹. Any molecules of UmuD that escape proteolysis need to be post-translationally processed by cleavage of the N-terminal 24 amino acids to a mutagenically active form, UmuD' ⁶⁰. Both UmuD and UmuD' form homodimers, but preferentially heterodimerise and in the heterodimer context, UmuD' is specifically degraded by the ClpXP protease⁵⁹. Thus, mutagenically active homodimeric UmuD' only accumulates in a cell after severe DNA damage. This delays the appearance of error-prone polV (UmuD’₂C) until ~45 mins after the initial DNA damage, thereby allowing the cell time to repair the damage via error-free repair mechanisms, before utilizing the error-prone TLS polymerase to traverse the damage.

In comparison, transcriptional and translational regulation of Y-family polymerases in eukaryotes is less coordinated and appears not to play a central role as in E. coli. One exception is the profound cell cycle regulation of Rev1 in S. cerevisiae. Rev1 levels increase c. 50-fold during G2⁶¹. This is achieved substantially at the level of protein stability, REV1 transcript varying by no more than three-fold between G1 and G2^{61, 62}. However, it is again likely that controlling the number of polymerase molecules provides only a crude degree of regulation and that low levels do not preclude activity.

Sub-cellular concentrations at [OK? Yes]  replication foci

A further refinement to the regulation of polymerase levels is to concentrate the enzymes locally in the vicinity of distressed replication forks. This approach appears particularly important to vertebrate cells in which the polymerases accumulate in sub-nuclear foci, visible by either indirect immunofluorescence or by fluorescent protein tagging, following DNA damage (Figure 5B). These foci, which have been termed ‘replication factories’, are thought to contain multiple replication forks, including forks stalled by DNA damage^{63, 64}. Over the past few years sub-nuclear focus formation has been increasingly used as a surrogate for enzyme function. However, the link between the two is far from clear. For instance, mutations in the C-terminal motifs of Polη (see Fig 4 and below) drastically reduce its accumulation in foci, while having rather minimal effects on the ability of the protein to complement the UV sensitivity of an XPV (Polη-deficient) cell line⁶⁵. This suggests that there may be two stages to the recruitment of the Y-family polymerases. The first promotes an increase in the local concentration of the enzyme in the vicinity of distressed replication forks, while the second, discussed in the following section, results in the selection and loading of the polymerase onto the end of the growing DNA strand to initiate TLS. These two processes are interdependent but distinct and we believe that this distinction is conceptually helpful, as we examine more specific mechanisms that are likely to control polymerase access to a particular primer terminus, be it at a replication fork, or at a post-replicative gap.

Regulation at stalled forks

As already noted, the relatively simple ‘mass action’ approaches to regulating the Y-family polymerases are unlikely to provide sufficiently tight regulation to be safe. Even formation of high local concentrations within replication factories is likely to be unsatisfactory on its own, as each ‘factory’ is a large structure and contains multiple forks (Figure 5B). Consequently it is also necessary to have more specific signals indicating which replication forks require assistance  (Figure 5C). A common feature of impeded replication is the exposure of single-stranded DNA near the stalled fork, and in both prokaryotes and eukaryotes this is likely to be the common denominator for signalling replication distress. While the mechanisms linking single-stranded DNA to polymerase activation are quite distinct in E. coli and in eukaryotes, they achieve the same end, namely the focussing of translesion polymerase activity to where it is needed.

Access to the clamp and PCNA ubiquitination

Fully effective activity by all DNA polymerases requires them to be tethered to the DNA through interaction with the sliding clamp, (b-clamp in prokaryotes, PCNA in eukaryotes) (See Figure 1 and 3). Eukaryotes appear to have exploited this interaction as a mechanism for regulating which type of polymerase, replicative or translesion, has access to the primer terminus. Many proteins potentially interact with PCNA via a motif designated as a PIP box⁶⁶ and pols η, ι and κ all possess PIP box motifs in their non-catalytic C-terminal extensions (Figure 4). The control of which enzymes are bound to PCNA at the primer terminus is partly determined by their widely differing association and dissociation constants, but further specificity is provided by post-translational modification of PCNA itself, principally by ubiquitin and SUMO⁶⁷. Thus, regulation of these modifications is likely to contribute to controlling the interaction of PCNA with potential client proteins, including the Y-family polymerases.

Replication arrest leads to the exposure of single stranded DNA, and PCNA mono-ubiquitination provides a key link between this event and the recruitment of the TLS polymerases. PCNA mono-ubiquitination is principally mediated by the E3 ubiquitin ligase Rad18 acting in concert with the E2 enzyme Rad6^68-71, although a number of other proteins have also now been implicated in creation of this modification^{72, 73}. Crucially, the activity of RAD18 in ubiquitinating PCNA is stimulated by its association with single-stranded DNA coated with the single-stranded DNA binding protein, Replication Protein A (RPA)⁷⁴.

As well as the PIP boxes, all eukaryotic Y-family polymerases have UBM or UBZ ubiquitin-binding motifs (Figure 4), which increase their affinity for ubiquitinated PCNA at the site of blocked replication^75-77, and thereby increase the probability of their being employed to replicate the lesion. The use of the term ‘probability’ in this context is important. Recruitment of polymerases into foci, and presumably also to the primer terminus, is highly dynamic. For polh and poli, the residence time in sub-nuclear foci is less than one second and what appears to be modulated by ubiquitin binding, for instance, is the residence time of individual molecules in the vicinity of the fork⁷⁸.

Although PCNA ubiquitination is central to regulation of translesion synthesis, evidence from chicken DT40 cells and mice carrying a K164R mutation in PCNA has revealed that a component of TLS is independent of PCNA ubiquitination in vertebrates ^79-82. As discussed below, at least in DT40, this PCNA ubiquitination-independent TLS appears largely dependent on Rev1 ^{81, 83}.

The role of Rev1

Rev1 plays an additional key non-catalytic role in the control of translesion synthesis, that in vertebrates is at least in part independent of PCNA ubiquitination ^{81, 83}.  The last 100 or so amino acids of vertebrate Rev1 binds pols κ, η and ι^84-86, as well as Polζ through its Rev7 subunit⁸⁷ and disruption of this domain leads to an effectively null phenotype⁵¹. Rev1 also interacts with PCNA, suggesting that its role could be as an adaptor between the clamp and the other polymerases, although there is some dispute about which sequences are involved in its interaction with PCNA^{51, 88-90}. The role of the two UBMs of Rev1 is enigmatic. The Rev1 UBMs bind ubiquitin and one of them is needed for cell survival and mutagenesis following DNA damage⁹¹, but PCNA ubiquitination is not required for Rev1 function at the replication fork⁸³. This structural role of Rev1 in coordinating TLS polymerases with PCNA appears to be conserved in S. cerevisiae although the way in which it interacts with polη/Rad30 and Rev7 is different^{90, 92, 93} (Figure 4).

Post-translational modifications

An additional level of complexity is introduced by the observation that the Y-family polymerases can themselves be post-translationally modified by ubiquitination, phosphorylation and possibly SUMOylation^{75, 91, 94-97}. The best characterised of these modifications is ubiquitination of Polη (Figure 5A). Polη is ubiquitinated in its C-terminus⁹⁵, by the E3 ubiquitin ligase, Pirh2⁹⁸ and, interestingly, this ubiquitination inhibits its interaction with PCNA^{95, 98}. Polη ubiquitination is reduced following DNA damage, suggesting an additional level of control of Polη recruitment. It remains to be determined whether this mode of regulation applies to other Y-family polymerases but, in addition, there are reports of damage-induced phosphorylation of Rev1 in S. cerevisiae^{94, 96}, and of polh in human cells⁹⁷. While the biological importance of the former remains to be established, the latter phosphorylation has a clear role in facilitating TLS⁹⁷. Taken together, these findings suggest that the regulatory mechanisms of these enzymes are rather more complex than initially thought. Indeed, there are reports of a number of other proteins that have been implicated in the recruitment of the Y-family polymerase to the replication fork^99-103 and in regulating the ubiquitination of PCNA^104-107.

Polymerase selection

Despite this complexity it seems that a basic principle for the recruitment of the Y-family polymerases to sites of arrested replication is control of the affinity of the enzymes for PCNA, or other structures near the fork, through modulation of the number of points of contact. None of these recruitment mechanisms, however, really provides any clear specificity in terms of which polymerase is recruited to a specific lesion. This remains an important question. One obvious selector is the lesion itself. It is self-evident that that once a set of polymerases is recruited to the vicinity of an arrested fork, a polymerase can only be utilised if it can accommodate the damaged template-primer in its active site and carry out a catalytic step before dissociating. Selection will thus be determined by normal enzyme kinetic parameters. The various levels of regulation are summarised schematically in Figure 5.

Timing of lesion bypass

It has been debated for many years whether TLS occurs at the replication fork or whether gaps are left to be sealed post-replicatively behind the fork. Early experiments in both E. coli² and mammalian cells¹⁰⁸ suggested the latter and indeed gave rise to the term post-replication repair to describe the process.  With the discovery of the Y-family polymerases, there was an assumption that they acted at the replication fork, though this has not been shown experimentally. More recent data have provided direct visual evidence for the existence of post-replicative gaps behind the forks in UV-irradiated S. cerevisiae¹⁰⁹ and elegant genetic studies, in which either Rad18, Rev3 or Polh were induced only in G2, have indicated that TLS can actually occur in G2 after the bulk of the DNA has been replicated^110,111. It is evident therefore, that TLS can occur post-replicatively. Although there are likely to be topological differences between TLS at or behind the fork, this does not necessarily imply major mechanistic differences. TLS at the fork requires that it occurs before the replicative helicase has run sufficiently far ahead to allow downstream re-initiation beyond the lesion, whereas the converse is true for TLS behind the fork. The differences may merely be kinetic, depending on which of these processes occurs first, and this will in turn be dependent on the factors that we have described in the previous sections. On the other hand, genetic studies in UV-irradiated chicken DT40 cells have suggested that Rev1 is involved in promoting TLS at the fork, whereas PCNA ubiquitination is required behind the fork (Figure 5C), suggesting that there are likely to be subtle mechanistic as well as kinetic differences ⁸³, a suggestion supported by a recent study showing altered mutation spectra originating from TLS taking place in S phase as opposed to G2 in mammalian cells ¹¹²

‘Non canonical’ roles

In addition to their roles in the replication of damaged DNA, Y-family polymerases have also been co-opted into a number of other related processes (Figure 6). [Bullet points are not within the style of the journals. Could you please modify the section to avoid them? A possibility could be to introduce there that five non-canonical roles have been identified, and then list them as First, second, etc.]

During development of the immune response, the antibody genes of vertebrates exhibit a particularly high rate of focussed mutagenesis, known as somatic hypermutation, which is driven by Activation Induced Deaminase (AID) ¹¹³. Although AID can only deaminate dC to dU, its action gives rise to mutations at all four bases in a series of reactions that critically depend on the Y-family polymerases ¹¹⁴. The dU formed by the action of AID is removed by Uracil DNA Glycosylase (UNG) resulting in an abasic site. Direct replication of this abasic site involves Rev1 and generates mutations at dG/dC basepairs ^{52, 54, 55}. Recognition of dU can also result in the formation of a single strand gap and the filling of these gaps by polh results in mutations at dA/dT basepairs ^{115, 116} (Figure 6A).

Other examples of non-canonical roles occur during excision repair processes. Clusters of damaged bases pose a particular problem to cells as excision repair can be affected by adjacent base lesions. Polh has recently been shown to play a role in the repair synthesis of clustered oxidative lesions¹¹⁷ (Figure 6B). Nucleotide excision repair is the central mechanism by which helix-distorting base lesions, including CPDs are removed. It involves the recognition and excision of a single strand tract of DNA containing the damaged base, followed by resynthesis using the undamaged strand as a template. Previously, it had been assumed that this synthesis was carried out by the replicative polymerases, but recently Polk was also shown to be involved particularly during conditions of low deoxyribonucleotide pools ¹¹⁸ (Figure 6C).

Further involvement of Y-family polymerases in non-canonical roles include the requirement for Rev1 alongside specialised DNA helicases for replication of DNA capable of forming a particular secondary structure, the G quadruplex, which can block replicative DNA synthesis ^{119, 120}. Additionally polh and polk have roles in replicating past unusual structures¹²¹, especially at fragile sites¹²² (Figure 6D). Finally, there is evidence for a role for polh in some forms of homologous recombination^{123, 124} (Figure 6E).