Cells possess many DNAPs; for example, human cells, yeast (S. cerevisiae) and E. coli have at least fifteen, eight and five, respectively, [6–22]. Most TLS-DNAPs are in the Y-Family [6–22], where humans have three template-directed members (hDNAPs η, ι, and κ), yeast has one (scDNAP η), and E. coli has two (ecDNAPs IV and V). Y-Family DNAPs have a conserved ~350aa core, which includes the polymerase active site (representative references [23–40]). As with all DNA polymerases, Y-Family members resemble a right-hand with thumb, palm, and fingers domains, although their “stubby” fingers and thumb result in more solvent accessible surface around the template/dNTP binding pocket [19], which is undoubtedly the case to accommodate the bulky and/or deforming DNA adducts/lesions that protrude into these open spaces during bypass. Y-Family DNAPs grip DNA with an additional domain, which is usually called the “little finger domain” or the “polymerase-associated domain” (PAD) [23–25].

Y-Family DNAPs are found in all three domains of life, bacteria, archaea, and eukaryotes, which undoubtedly reflects the fact that all cells face the same issues when confronting the need to replicate past DNA damage. The pattern of TLS is often strikingly similar in different cell types. For example, human DNAP κ was originally discovered because its sequence closely resembles E. coli DNAP IV [41–43], and dNTP insertion opposite a variety of adducts/lesions is remarkably similar for the DNAP IV/κ pair (Table 1), suggesting they are functional orthologs (discussed in [44]). E. coli DNAP V and human DNAP η are also functional orthologs, based on their similarity of dNTP insertion opposite a variety of adducts/lesions (Table 1, [44]). Cases have been made that the IV/κ-class is present in cells to bypass endogenously generated N²-dG adducts, and the V/η-class is present to bypass UV-induced photoproducts, as discussed below.

B-Family DNAPs can also be involved in TLS, such as DNAP II in E. coli and REV3 (the polymerase subunit of DNAP ζ), which is present in most eukaryotes [6, 7, 13–15]. B-family TLS-DNAPs are involved in a DNA repair process involving some interstrand DNA cross-links [45–49] and in TLS of some adduct/lesions (see below).

Although DNAP V replicates undamaged templates with relatively low fidelity (10⁻³ to 10⁻⁴) [54], one striking quality is its ability to accurately bypass UV photoproducts; for example, it inserts dATP opposite TT-CPDs [54]. Analysis of insertion tendencies opposite a variety of adducts/lesions led to the observation that DNAP V may have two insertion modes: (i) correct dNTP insertion, and (ii) default dATP insertion [44]. UV light is a frequently encountered form of DNA damage for which a TLS-DNAP might be important, and since TT-CPDs are the major UV lesion [55], a default dATP insertion mode might help minimize UV mutagenesis. However, the utilization of this second mode in other circumstances may have drawbacks. For example, UV mutagenesis also depends on the umuD/C genes, implying that DNAP V is required for UV mutagenesis,where C→T mutations in 5′-PyC sequences predominate, which also implies dATP insertion (discussed in reference [56]). DNAP V is involved in other mutagenesis pathways; for example, it inserts dATP opposite +BP in the G → T mutational pathway [57], as discussed below. In fact, the preferential mutagenic insertion of dATP opposite a variety of DNA lesions in E. coli has been called the “A-rule” (see [58, 59] and references therein), and it seems likely that this is attributable to DNAP V's tendency to insert dATP [44]. Based on lesion-bypass specificity (Table 1), E. coli DNAP V appears to be the functional ortholog of human DNAP η [36], which is almost certainly responsible for correct bypass of UV-lesions in human cells and minimizing UV-light mutagenesis that leads to skin cancer [60–65].

On its own, UmuC, which is the polymerase subunit of DNAP V, either misfolds or aggregates and is found in inclusion bodies [22, 50, 66]. UmuC copurifies with UmuD', though the yield is invariably low [22, 50, 66]. RecA is also required for efficient DNAP V activity, and recently, the “DNAP V mutasome” was shown to be a UmuC/UmuD'₂/RecA heterotetramer [67, 68]. The RecA monomer is added from the 3′-end of a RecA filament either in cis or in trans, where the former seems intuitively more likely, since UmuC/UmuD'₂ would encounter a 3′-cis-RecA at a lesion site, given that RecA filaments coat ss-DNA on the downstream side of a lesion-blocked replication fork. To form RecA filaments on ss-DNA, SSB must first be removed, which is accomplished by RecFOR [69]. Interestingly, some evidence suggests that the RecA eukaryotic homolog Rad51 is able to stimulate DNAP η, which is the DNAP V ortholog [70]. β-clamp also plays a significant role with DNAP V as discussed below.

DNAP IV replicates undamaged DNA only ~5-fold less accurately than the catalytic α-subunit of DNAP III [54]. It is prone to making −1 frameshift mutations in homopolymeric runs of six or more G:C base pairs, and base substitutions also result [22, 71]. DNAP IV's most striking quality is its ability to accurately bypass a variety of N²-dG adducts [72–77]. Methylglyoxal is produced nonenzymatically from various cellular trioses and forms N²-(1-carboxyethyl)-2′-dG as its major stable adduct, which is bypassed accurately by DNAP IV [76]. Oxidative metabolism forms reactive oxygen species that generate lipid peroxidation products that give exocyclic adducts, some of which can ring-open to N²-dG adducts in ds-DNA [78] and might be bypassed by DNAP IV, though this has not been investigated experimentally. These observations have led several groups to speculate that the cellular rationale for the genesis of the IV/κ-class of Y-Family DNAPs is the accurate bypass of N²-dG adducts derived from various endogenous mechanisms [72, 76].

No analogous story vis-a-vis adducts/lesions has yet emerged to provide a rationale for the presence of B-family DNAP II in cells, though one possibility is its involvement in an accurate DNA repair pathway for interstrand cross-links [45]. An analogous pathway involving B-family DNAP ξ exists in eukaryotic cells, and a pathway has been proposed [46–49]. As discussed below, DNAP II functions in other TLS pathways.

UmuD₂C (not UmuD'₂C) is thought to slow down normal DNA replication in response to DNA damage, thus allowing additional time for lesion removal, which is considered a DNA damage checkpoint analogous to what happens in eukaryotic cells [79]. Another mechanism to accomplish this was recently described: DNAP II or IV can associate with the DnaB helicase and slow down the replication fork [80].

REV1 is not a traditional template-directed polymerase and does not use base-base hydrogen bonding. Rather REV1 is a dCTP insertase that flips template dGs out of the helix, after which dCTP insertion is directed by hydrogen bonding to a REV1 arginine residue [83]. REV1 seems to play a central role in many lesion bypass events as a structural component, and DNAPs κ, η, and ι each have REV1 binding domains [9].

DNAP κ is the eukaryotic ortholog of DNAP IV (Table 1), and they seem to be present in cells to accurately insert opposite N²-dG adducts [84], for example, DNAP κ deficient cells are sensitized to killing by benzo[a]pyrene, which predominantly forms an N²-dG adducts [85]. DNAP κ uniquely has an N-terminal extension of 100aa called the “N-clasp” [35]. The N-clasp has three α-helices in a U-shape, one of which (~aa30–50) binds on the surface of the fingers domain, the second of which (~aa50–75) links the fingers and thumb domains and lies diagonally across the duplex region of DNA, and the third traverses the thumb domain to the usual site of the N-terminus in Y-Family DNAPs. Removal of the N-clasp significantly decreases DNAP κ polymerase activity [35]. The presence of the N-clasp has implications for lesion bypass; for example, DNAP κ does not bypass the N⁶-dA adduct of benzo[a]pyrene [86], which has been attributed to a steric clash between the N-clasp and the pyrene moiety, as revealed in a molecular modeling study [87]. DNAP κ structure is considered below.

A major role of DNAP η is nonmutagenic bypass of UV lesions, such as TT-CPDs, and humans deficient in DNAP η have the cancer-prone syndrome Xeroderma pigmentosum variant (XPV), which leads to a high incidence of UV-induced skin cancer [60–65]. Both human and yeast DNAP η preferentially insert dATP opposite the 5′-T and 3′-T of a TT-CPD, with misinsertion being higher at the 3′-T, where dGTP is incorporated ~3% of the time [88, 89]. Recently, X-ray structures of a TT-CPD in the active site of yeast DNAP η [39] and human DNAP η [40] have emerged. These findings are presented in a separate section(Section 8),after certain principles about Y-Family DNAPs structure have been discussed. DNAP η also plays a role in accurate bypass of the oxidative lesion 8oxoG [90] and adducts formed by the anticancer drug cis-platinum [91].

The role of DNAP ι in cells is more enigmatic, though the fact that deficient cells show enhanced sensitivity to oxidative damage may be revealing [92]. One interesting feature of DNAP ι is its propensity to use syn-purines in the template to form Hoogsteen base pairs syn-A:T and syn-G:C[36, 37].A cellular rationale for this is the following. Oxidative damage leads to lipid peroxidation products,which form exocyclic adducts that block the Watson-Crick antiface of the DNA bases. Exocyclic purine adducts in the syn-configuration can still base pair through their Hoogsteen face. The syn-A:T base pair has two hydrogen bonds, just like anti-A:T. However, both syn-G:C and syn-G:T base pairs have only one hydrogen bond, unless a proton is trapped. In a syn-GH⁺:C base pair the trapped proton is between N7G and N3C, whose pKa values are relatively high (~3), while a trapped proton in a syn-GH⁺:T base pair would be between O⁶G and O⁴T, whose pKa values are much lower. Thus, a syn-GH⁺:T base pair is expected to be less stable than a syn-GH⁺:C base. Evidence for DNAP ι using syn-GH⁺:C base pairing exists [37]. 1,N⁶-etheno-A directs both dCTP and dTTP incorporation, and in this case both require a trapped proton [38], where the pKa values of the relevant atoms trapping the proton are more equal. While such thinking is considered satisfying [12], one finding suggests that the situation can be more complex. DNAP ι preferentially incorporates dCTP opposite the major adduct of 2-acetylaminofluorene (AAF-C8-dG) [93], Syn-AAF-C8-dG places the bulky AAF-moiety in the minor groove, where molecular modeling showed that it does not fit, while anti-AAF-C8-dG:C pairing, which places the AAF-moiety in the spacious major groove side of DNAP ι, is possible [94]. The authors propose that purine adducts with bulk on the minor groove side probably use syn-purine pairing, but that purine adducts with bulk on the major groove side probably use antipurine pairing. Anti-AAF-C8-dG:C pairing requires a modest change in sugar pucker (from C3′-endo to C1′-exo), as noted in modeling studies with both DNAP ι [94] and Dpo4 [95].

Upon DNA binding to Apo-Dpo4, the thumb/palm/fingers domains do not change their structure dramatically. However, the little finger domain acts like a door, which is open in Apo-Dpo4, and then rotates ~130° to close around DNA; in particular, it binds in the major groove in the duplex region from about L + 3 to L + 8 [33]. This motion is facilitated by the fact that the little finger is connected to the rest of the protein by a simple ten amino acid tether. Once binary-Dpo4 is formed, the palm, fingers and little finger translate ~3.3Å along the helix as the next template base slides into the active site, which opens the space into which the complementary dNTP binds to give ternary-Dpo4 [32]. The thumb domain, however, does not move in this step, but, rather, moves either before, during, or after the subsequent covalent reaction step. A variety of subtler changes in Dpo4 structure are also reported to accompany these steps [32, 33]. Kinetic studies reveal that Y-Family DNAPs have a rate-determining conformational change before dNTP incorporation [10, 96], and three conformational states E, E′, and E′′ have been reported, where the E′ → E′′ conformational transition is rate determining, though the nature of these states have not been identified. Recently, hydrogen-deuterium exchange in tandem with mass spectrometry has been used to study conformational changes in Dpo4 brought about by dNTP binding [30]. Correct dNTP binding affects the structure of a loop between the B-helix and the C-helix above the Dpo4 active site. (The positioning of these features can be inferred from the UmuC(V) sequence in Figure 2.) Another conformational change was also detected in the H-helix, which contacts the primer strand and was proposed to move away from the active site in conjunction with ds-DNA movement to permit room for correct dNTP binding. The F-helix also moves, but this motion is not specific for the correct dNTP. In terms of lesion bypass, Dpo4 showed decreased catalytic efficiency with increasing bulk of N²-dG adducts, which was attributed more to the effects of the bulky lesion on the rate of the catalytic step than on the rate of the conformational steps [31].

Several studies have shown that dNTP incorporation is more dependent on base:base hydrogen bonding for Y-Family DNAPs than for DNAPs in other families during the replication of both undamaged and damaged DNA [97–99].

DNAP IV binds β-clamp to help release a stalled DNAP III from the same β-clamp, leaving DNAP IV/β-clamp at the site of the lesion [110]. This process is rapid (t < 15s). Presumably, a similar mechanism operates for each TLS-DNAP (II, IV and V), which all have β-clamp binding sites (consensus: QLxLF) that are required for them to be active in E. coli [111]. An X-ray structure shows that the underlined amino acids QLVLGL at the C-terminus of DNAP IV form the main interactions with a “cleft” in the β-clamp [112]. The α-subunit of DNAP III and the δ-subunit of the γ-complex also bind to the cleft in the β-clamp. DNAP IV and V can also bind to a site in the “rim” of the β-clamp, but this seems unimportant for TLS [113–115]. In vitro studies show that β-clamp stimulates both polymerase activity and processivity of TLS-DNAPs: the addition of β-clamp in vitro increases DNAP IV activity ~2000-fold and processivity from 1 nucleotide to ~400 nucleotides, and also increases DNAP V activity ~100-fold and processivity from 1-2 nucleotides to ~18 nucleotides [22].

What factors affect the choice about which TLS-DNAP will insert opposite a particular lesion? Several lines of evidence suggest that E. coli has a hierarchy for the replication of normal, unadducted DNA when DNAP III is inactivated: DNAP II > IV > V [116]. (The assays did not permit an assessment of DNAP I.) Since this order (III > II > IV > V) does not reflect the relative concentration of these DNAPs in cells (see above), another mechanism for decision making was suggested, such as relative DNAP affinity for the β-clamp. This order does reflect relative fidelity of these DNAPs and would be a sensible order for E. coli to allow TLS-DNAPs to initially sample adducts/lesions prior to a decision about which will do TLS. But the ultimate decision is probably predominantly controlled by which TLS-DNAP is most efficient at bypassing a particular adduct/lesion biochemically.

AAF was originally developed as a potential pesticide, but it was abandoned when it was found to be a potent rat carcinogen [118]. Following activation, AAF principally binds at C8-dG, as do most aromatic amine mutagens/carcinogens, where AAF and AAF-C8-dG have frequently been used as models to probe the mutagenic and carcinogenic mechanisms of aromatic amines [117]. In E. coli AAF has a major mutational hot spot in 5′-CG₁CG₂ sequences in which it induces −2 frameshift mutations [117]. AAF-C8-dG at G₂ (but not G₁) causes a −2 frameshift mutation in a DNAP II-dependent process, or causes no mutation in a DNAP V-dependent process [117]. The current model is that AAF-C8-dG at a replication fork exists in two different conformations [66, 117]. In one conformation, the adducted dG moiety is in a −2 slipped intermediate, which DNAP II uses for insertion, and then in the presence of β-clamp an additional three extension steps (to L + 3) are accomplished, at which point replication can be successfully continued by DNAP III [66]. From a nonslipped intermediate, DNAP V inserts dCTP opposite AAF-C8-dG and then extends by adding four more dNTPs (to L + 4), after which DNAP III can successfully continue replication [66]. These two pathways are followed approximately equally in cells, though by manipulating the concentration of DNAP II versus DNAP IV, the ratio [−2 frameshift:no mutation] can be modulated, suggesting that the two conformations interconvert [93]. In vitro in 5′-CG₁CG₂ sequences DNAP II also does TLS to give a bypass product that should ultimately yield a −1 frameshift mutation, which are not, however, observed in vivo; recent in vitro studies suggest that DNAP II cannot extend far enough from the −1 frameshift intermediate, and, thus, the 3′ → 5′ exonuclease activity of DNAP III degrades the intermediates in the −1 frameshift pathway [66].

Molecular modeling has provided insights about how lesion bypass might occur; for example, a modest alteration in sugar pucker (from C3′-endo to C1′-exo) is required before AAF-C8-dG can Watson-Crick base pair with dCTP [94, 119]. Though this work was done in Dpo4 and hDNAP ι, there is every reason to think that a similar conclusion would be reached for DNAP V. Recently, X-ray structures of the corresponding deacetylated adduct AF-C8-dG has been reported [32]. Regrettably, the structures do not reveal insights about how dCTP might be inserted opposite AF-C8-dG, but they do offer a glimpse of more-or-less normal Watson-Crick AF-C8-dG:dC base pairing in the L + 1, which has the AF moiety in the opening on the major groove side of Dpo4, and in the L + 2 position, in which the AF-moiety is accommodated by a modest rearrangement in the little finger domain.

Benzo[a]pyrene (B[a]P) is a well-studied DNA damaging agent that is a potent mutagen/carcinogen and an example of a polycyclic aromatic hydrocarbon (PAH), a class of ubiquitous environmental substances produced by incomplete combustion [120, 121]. PAHs in general and B[a]P in particular induce the kinds of mutations thought to be relevant to carcinogenesis and may be important in human cancer [122–128]. B[a]P mutational spectra were established with the major metabolite that reacts with DNA (i.e., (+)-anti-B[a]PDE), in E. coli [129], yeast [130, 131] and mammalian (CHO) cells [132]. Mutagenesis has also been studied with [+ta]-B[a]P-N²-dG (+BP, Figure 1), the major adduct of (+)-anti-B[a]PDE, and G → T mutations predominate in most cases (see [133] and references therein).

A variety of architectural features are revealed by considering how B[a]P-N²-dG adducts must sit in the active sites of Y-family DNAPs and how these structures might relate to adduct processing [136]. To form an adduct-dG:dCTP base pair, the B[a]P moiety must be in the developing minor groove, since the adduction site (N²-dG) is in the minor groove in a Watson-Crick base pair. On the minor groove side, Y-Family DNAPs have an opening (or gap) next to the active site between the fingers and little finger domains. This opening looks like an elliptical hole of varying sizes in Dpo4 [24–32], Dbh [23], hDNAP ι [36–38] and in models of DNAP IV and UmuC(V) [44], while it looks like a slot in hDNAP κ [35]. It is not unreasonable to think that the size and shape of this opening might influence dNTP insertional mechanism given that the bulky B[a]P moiety must interact with this opening on the minor groove side.

The character of this opening can be analyzed based on a simple analogy to a “chimney.” Three regions of the protein contribute to the chimney as shown in Figure 3(a) for our model of DNAP IV: an upper lip (aa33–36, turquoise) and a left lip (aa73–76, blue), which are in the fingers domain, while the lower lip (blue, aa244–247), is in the little finger domain [136]. The UmuC(V) chimney is shown in Figure 3(b).

Two features control the size and character of this opening. (1) The amino acid side chains in the upper lip (aa33–36 in DNAP IV) can be thought of as a “flue,” which either plug the chimney leaving a small opening or do not plug the chimney leaving a large opening. The flue amino acids are present in all Y-Family DNAPs. (2) A “cap” may lie over the top of the chimney opening. The cap is formed by an insert of amino acids in the left lip of the chimney and is only preset in DNAP η [39, 40].

First, we consider how the “flue” amino acid side chains influence the character of the “chimney.” Why do we think that the chimney is a key structural feature that is likely to be important for protein function? If the chimney is important, then evidence for its importance should exist, even in the case of UmuC where no X-ray structure exists. We aligned 408 UmuC(V) sequences, and Figure 2 shows the total number of each of the twenty amino acids that are found at each aa position. Positions with ≥90% aa homology or with clusters of high homology are highlighted in pink and red. Many of the first ~20aa show high homology, including the presumptive catalytically essential aspartate (D6), and the steric gate (Y11). The region around the catalytically essential asparate/glutamate pair (D101/E102) is also highly conserved. These and other regions that are conserved in all Y-family DNAPs are highlighted in pink in Figure 2. Regions conserved in UmuC(V), but not in other Y-Family DNAPs, are highlighted in red in Figure 2. One such conserved region is V29-C36, which is part of a loop that includes one edge of the chimney lip (S31-D34). (This loop is discussed at greater length in Section 7.1). A second conserved region is S71-Y77, which includes the second lip of the chimney, as well as other features discussed below. This conservation is strong evidence that the nature of the chimney opening is important. The third edge of the chimney (E255-T258) is in the little finger domain, and in our model of UmuC the third lip is farther from the active site and appears less likely to impinge on adducts protruding from the minor groove. Consistent with this view the third lip of the chimney is less well conserved. Preliminary analysis of the chimney lips of large collections of DNAPs IV, κ, and η sequences also reveal considerable amino acid conservation of the chimney lips.

dATP adopts the canonical chair-like shape found in all families of DNAPs (see Section 10),though the shape is nuanced; for example, the angle of the A-base is tilted slightly downward compared to other dNTPs in Y-Family DNAPs in order to pair with the 3′-T of the TT-CPD. In hDNAP η, the guanidinium of R61 interacts with phosphate-oxygens on both the α- and β-positions of dATP, which is a unique interaction among Y-Family DNAPs. Interestingly, the equivalent R73 in yDNAP η is flexible and can be in this position, or it can face the opposite direction and pair with the extra template base in the active site; that is, the base not paired with the dNTP. This arginine is one of the most conserved amino acids in DNAP η, though the R73A mutation in yDNAP η retains normal kinetics with respect to both undamaged and damaged DNA, which suggests that its most important role is not being revealed in studies with a TT-CPD. In UmuC(V), which is the bacterial ortholog of DNAP η, methionine (M51) is usually at the equivalent position, though arginine or lysine are frequently present (42/408) (Figure 2). The other highly conserved amino acid in DNAP η is a glutamine (Q38 in hDNAP η and Q55 in yDNAP η), which sits in the minor groove and interacts with both O²-positions on the T-bases of the TT-CPD.

Figure 4 shows the correct alignment based on the X-ray structures (as described in the legend), including the little finger domain of DNAP IV [112]. What features of these sequences allow the structures to be conserved, even though their primary amino acid sequences are not conserved? An inspection of the X-ray structures reveals that little finger domains are held together by a core of about twenty-one hydrophobic residues, which are highlighted in turquoise in the alignment in Figure 4 (H1–H21) and are shown in a Dpo4 structure (Figure 5). Though hydrophobicity is conserved at these twenty-one positions, the exact amino acid is not. Dpo4, Dbh, DNAP κ, DNAP IV, DNAP ι, yDNAP η, and hDNAP η have 19, 19, 17, 20, 20, 20, and 20 hydrophobic residues, respectively, at these 21 positions (Figure 4).

A comparison of these seven proteins reveals that the little finger domain has thirteen positions where an amino acid side-chain can interact with a phosphate-oxygen. Nine positions have a consensus lysine, arginine, asparagine or glutamine, which can interact with a phosphate-oxygen in the DNA backbone; they are designated L1–L9 in the alignment in Figure 4 (red) to indicate that their R-groups are “long.” They are also shown in the Dpo4 structure in Figure 5 (red). Four positions have a consensus serine or threonine that can interact with a phosphate-oxygen; they are designated S1–S4 to indicate that their R-groups are “short” in Figure 4 (pink). They are also shown for Dpo4 in Figure 5 (pink).

In terms of DNA interactions, there are some nuances. In several cases amino acids with longer R-groups can also serve at the S1–S4 positions (e.g., K301 in Dbh). Regarding S3, S297/Dpo4, S297/Dbh and S359/DNAP ι clearly interact with the P + 6 phosphate-oxygen; however, T469/DNAP κ looks like a rotation would be required for it to interact properly, though it was counted as a positive. Q296 in DNAP IV might be able to interact with P + 5, though DNA is not present for definitive assessment and it is noncanonical, so it is not counted. R285/DNAP IV and R283/Dbh (instead of N340) might interact with P + 8, though DNA is not present for definitive assessment and it is non-canonical, so neither is counted.

In contrast, UmuC(V) does not give a satisfactory structure when +BP is paired with dCTP (Figure 3(b)/center), because UmuC(V)'s small chimney opening forces the bulky pyrene moiety downward. Asparagine-32 is the main problem, and its side chain plugs the UmuC(V) chimney leading to a clash with +BP. In the unadducted structure (Figure 3(b)/left), N32 adopts its lowest energy rotational conformer with respect to the Cα–Cβ bond. The presence of +BP leads to a rotation about the Cα–Cβ bond (Figure 3(b)/center); however, no other rotation can get N32 any farther out of the way. Consequently, UmuC(V)'s small chimney forces the +BP-dG in the template and its paired dCTP to move downward such that the primer-O3′/dCTP-Pα distance is elongated to ~5.0Å, which is a nonreaction-ready distance.

These observations provide a reasonable rationale for why DNAP IV preferentially does cellular dCTP insertion in cells: DNAP IV's large chimney opening permits a reasonable adduct-dG:dCTP structure with reaction-ready distances between primer-O3′ and Pα-dCTP.

If UmuC(V)'s small chimney enforces a non-reaction-ready distance between the primer-O3′/dNTP-Pα, then how could UmuC(V) insert any dNTP opposite +BP? Recently, we offered a hypothesis [136].

X-ray structures from all DNAP families show a canonical dNTP shape that has been called “chair-like,” and its structure from T7 DNA polymerase is shown in Figure 6 (blue insert), which is also observed in most of the X-ray structures of Y-Family DNAPs, including Dpo4 (“S1-dNTP shape,” Figure 6, green). However, a second non-canonical “goat-tail-like” shape (“S2-dNTP”, Figure 6, yellow) has also been observed [26]. The goat-tail-like S2-dNTP shape can lie lower down in the active site, and +BP paired with dCTP in the S2-dNTP shape allows the pyrene to lie comfortably under the small chimney opening of UmuC(V), which allows the primer-O3′/dNTP-Pα distance to be reaction-ready (~3.8Å).

Interestingly, the S2-dATP shape allows the syn-conformation, such that syn-dATP can pair with adduct-dG via a Hoogsteen base pair. In contrast, the syn-adenine base in the S1-dNTP shape has steric clashes with atoms in the deoxyribose and the α-phosphate. Adduct-dG:syn-dATP pairing in UmuC(V) gave reasonable structures with primer-O3′/dNTP-Pα distances of ~3.6Å [136]. The S2-dNTP shape appears to have accompanying protein components that should allow phosphoester bond making and breaking [136, 138].

Though the dislocation/templated mechanism is attractive for Dpo4, considerable evidence both in vitro and in vivo suggest that neither DNAP IV nor DNAP V follow a dislocation/templated mechanism with +BP, as discussed extensively in [136] and references therein.

Why might Dpo4 be different than DNAPs IV and V? Dpo4 is in the IV/κ-class, and it has a nonbulky roof-aa and roof-neighbor-aa [A44/A57], as expected for the IV/κ-class. However, Dpo4 has a very small chimney opening (discussed below), which is associated with for the V/η-class. Thus, Dpo4 is a hybrid with a roof similar to the IV/κ-class and a chimney similar to the V/η-class.

Dpo4 has a small chimney opening, because its bulky flue-handle (C31) causes downward curvature of the chimney upper lip and leads to a closed flue (V32) [136]. In fact, Dpo4's chimney is exceedingly blocked: (1) the V32 flue is inserted deeper into the chimney than, for example, the N32 flue of UmuC(V), and (2) M76, which is the second amino acid in Dpo4's left lip, also plugs the chimney. DNAP IV and UmuC(V) have non-bulky G74 and S72, respectively, in the position equivalent to M76 in Dpo4. Thus, the excessively plugged chimney of Dpo4 forces the pyrene moiety of +BP so far from the active site that base pairing via either S1-dCTP or S2-dCTP is impossible; consequently, both the pyrene and the dG moieties of +BP are forced to be extrahelical with consequence being that pairing cannot occur with its complementary dC [28].

As mentioned above, DNAPs η, IV, η, and ι have an appropriate amino acid at 9, 8, 7, and 8, respectively, of the thirteen sites that can interact with phosphate-oxygens (L1–L9 and S1–S4). In contrast, Dpo4 and Dbh conform 13/13 and 11/13, respectively, which undoubtedly reflects the need for more interactions with DNA given that they are from thermophilic bacteria and must operate at an elevated temperature. Thus, Dpo4 studied at 37°C, which is typical, may give results that do not reflect correctly on aspects of the mechanism of other Y-Family DNAPs, which have evolved to operate at 37°C.