Although very significant progress has been made in both structural analyses of many of the TLS pols and the range of damaged DNA substrates that these pols can bypass, in the majority of cases, the biological role of these enzymes can only be inferred by changes in efficiency and mutagenic rates of TLS in their absence. Little data have been gathered concerning the role of these enzymes in developmental biology, their tissue-specific expression and steady-state distribution in adult organisms, the mechanism(s) for recruitment to sites of DNA damage, and the processes that regulate their expressions in response to environmental toxicant exposures and/or endogenous stresses. In the case of pol ν, the initial characterizations have demonstrated a very limited repertoire of lesions that pol ν can efficiently bypass (5
). Although a cellular role of pol ν in TLS has not yet been established, its high frequency of misincorporation of dT opposite dG suggests that its expression would be tightly regulated. Thus, understanding the potential substrate specificity of pol ν may help to guide future investigations of its biological role in TLS and circumstances for induction or activation. In this regard, we demonstrated that although pol ν could carry out TLS past bulky major groove DNA lesions such as N6
-dA PCLs and N6
-dA ICLs, it failed to replicate DNAs containing chemically similar minor groove DNA lesions. Thus, the location of the lesion within DNA is one important structural determinant that influences the ability of pol ν to bypass the lesion.
We also discovered that pol ν could not efficiently bypass εdA, and we speculate that this may be either due to pol ν requirement for the Watson−Crick edge for efficient dA templating or structural heterogeneity of the lesion within the active site. Regarding the former possibility, pol ν may fail to catalyze efficient TLS past εdA because the Watson−Crick edge of this adduct is blocked by the modification. The majority of DNA pols utilize the geometry of the canonical Watson−Crick pairs for the nucleotide selection (19
), but some TLS pols utilize alternative mechanisms. For example, pol ι induces a syn
conformation on template purines, which results in a Hoogsteen base pairing with the correctly matched incoming nucleotide and enables bypass of lesions that disrupt the Watson−Crick edge but not the Hoogsteen edge (20
). The diminished capability of pol ν to bypass εdA strongly suggests that the accessibility of the Watson−Crick edge of the template dA is important for the efficient replication by this pol; thus, it is likely that pol ν-catalyzed polymerization involves the recognition of the Watson−Crick geometry.
With regard to structural heterogeneity of εdA adduct, NMR analyses have revealed that orientation of the base around the glycosydic bond is affected by identity of nucleotide opposite the lesion (21
). When paired with dT, the εdA is in a normal anti
conformation; however, the εdA base prefers the syn
conformation when placed opposite a mispaired dG. Additionally, the cocrystal structure of DNA containing an εdA bound to pol ι also revealed that the lesion adopted a syn
). Thus, εdA can adopt a syn
conformation depending on the base it is paired with, as well as interactions with the pol. There is a possibility that similar to pot ι, the adducted base is in a syn
conformation in the pol ν active site. However, it is unclear whether either orientation of the ε-modified base can be efficiently utilized for nucleotide insertion.
Intriguingly, pol ν also could not fully bypass a (+)-BPDE-dA. The NMR solution structure of this lesion opposite dT in duplex DNA showed complex conformational heterogeneity (23
). Computational studies using an NMR structure of duplex DNA where dT was placed opposite the modified dA have revealed that the glycosydic bond of (+)-BPDE-dA was in syn
equilibrium; therefore, the modified dA has a diminished capability to participate in Watson−Crick base pairing (24
). It is possible that the glycosydic bond of modified dA rotates and adopts a syn
conformation at the primer-template junction and therefore cannot participate in Watson−Crick base pairing. An alternative explanation may be that the structure of the active site of pol ν is significantly distorted by bulky aromatic ring system of this lesion. It has been shown that the BPDE moiety of this lesion is intercalated into the double helix, on the 3′ side of the adducted dA, and thus causes significant structural distortions at the active site of T7 pol (25
). Because the T7 pol is also a member of the A-family pols, the inability of pol ν to bypass this lesion may be due to the active site distortions, similar to those shown for T7 pol. With regard to the low fidelity synthesis past this lesion by pol ν, it has been shown that the adducted dA is displaced from the active site of T7 pol as a result of steric hindrance between the BPDE moiety and the primer template. Among the four dNTPs, dATP fits best into the dNTP binding pocket that is enlarged due to the displacement of adducted dA (25
). This structural change provides a likely explanation for the predominant misincorporation of dA by pol ν.
Recently, we reported a comparative study of the mutagenic consequences of identical lesions linked in the major groove via N6
-dA versus the minor groove linkage via N2
-dG during the replication of single-stranded pMS2 shuttle vectors in COS-7 cells (11
); these lesions were the same site-specific PCL4s substrates used in this investigation. These data revealed that DNA containing either lesion could be replicated, with the N2
-dG PCL4 being moderately mutagenic, while the N6
-dA PCL4 showed very low mutagenic potential. Although it is anticipated that the extremely bulky nature of the N6
-dA PCLs lesions would block replicative pols, we carried out in vitro replication assays using pol δ (Figure ). The primer extensions and single nucleotide incorporations showed some limited bypass of N6
-dA PCL12 at very high dNTP concentrations (100 μM) (Figure a), while the titration experiment with varying concentrations of dTTP (from 6.4 nM to 100 μM) revealed the severity of the blockage posed by this lesion for the pol δ-catalyzed nucleotide insertion (Figure b). Specifically, pol δ could incorporate dT opposite the ND dA at concentrations as low as 6.4 nM with ~8% of primers being extended, while even at 100 μM dTTP, only ~2% of primers were extended on the N6
-dA PCL12-containing substrate. These results suggest that although in vitro conditions could be manipulated to observe bypass of N6
-dA PCLs by pol δ, the block to replication is likely to occur when pol δ encounters such bulky lesions; therefore, specialized pols may be recruited to carry out TLS. The data presented herein suggest that pol ν is a likely candidate to perform TLS past a variety of major groove lesions. At present, the structural basis is unknown why pol ν could efficiently bypass PCL and ICL as large as 3025 Da but fails to bypass less bulky BPDE adduct. However, we speculate that the lesions that are readily bypassed by pol ν may have significant conformational flexibility and thus, can be accommodated without distorting DNA−enzyme interactions within the pol active site. Overall, our data suggest that since pol ν is capable of bypassing extremely large major (but not minor) groove lesions, it may play a role in TLS following toxicant challenges that result in the formation of such lesions in the cells.
Figure 6 Replication bypass of N6-dA peptide cross-link by yeast pol δ. (a) Single nucleotide incorporations and primer extensions were catalyzed by 0.5 nM yeast pol δ with 2 nM primer template containing ND or N6-dA PCL12 under standing start (more ...)