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  • br Common features of TdT

    2019-07-31


    Common features of TdT, pol μ and pol λ TdT, pol μ and pol λ remain in a closed conformation throughout their catalytic cycle, contrary to pol β [24•, 53, 54•]. One possible explanation for this observation is that they have traded fidelity (which requires open-to-closed transition) for a very tight binding of the DNA synapsis, a very fragile structure. One unique feature revealed when the first X-ray structure of TdT was solved [], is a specific Loop (Loop1), composed of 20 Cyanine 3-dCTP to (382-401), located between the β3 and β4 strands (Figure 1b), that prevents the binding of a 5′ overhang of the template strand. More than 30 structures of TdT (wild-type or mutants in different complexes) are available on PDB and in all of them, Loop1 adopts the same lariat-like conformation that prevents the binding of an uninterrupted template strand on TdT (Figure 2). Perhaps somewhat deceptively, in all known structures of pol μ, Loop1 (also about 20 amino acids long) is invisible in the electron density map, meaning that this region is disordered (Figure 2, Figure 3a). In pol λ, Loop1 is comparatively shorter (8aa), but still longer than in pol β (Figure 3). However, pol λ has an additional Loop, called Loop3, that, interestingly, is located precisely where another form of TdT resulting from alternative splicing has an insertion of 20 additional residues [55, 56] (Figure 3b) and where it is ideally placed to control bulges or insertions just before the in trans templating base [] (Figure 3a). Mutations experiments have consistently shown the importance of Loop1 for the substrate specificity not only in TdT [27••, 57•] but also in pol μ [58•, 59, 60•], as well as pol λ []. Early sequence comparisons in a structural context helped to define two important regions for the specificity of TdT versus pol μ [], later named SD1 and SD2 [] (Figure 3): they are located at the C-terminal border of Loop1, and in a β-turn-β structure close to Loop1, which can also bind an extra Zn++ ion [] but the precise role of this additional divalent ion is currently unknown. Mutations in these two regions profoundly affect the activity of both TdT [27••, 28••] and pol μ []. Mutation of only one amino acid in SD1 region (F401A) confers to Tdt an in cis templated polymerase activity, even in the presence of Co++ [].
    Nucleotidyltransferase activity of TdT Extensive biochemical experiments have demonstrated that TdT can add random deoxyribonucleotides (dNTPs) on a ssDNA primer, which has to be at least three nucleotides long, in a template-independent manner [2]. In vitro experiments show that TdT can use all four natural dNTPs with a preferential incorporation of dCTP and dGTP compared to dATP and dTTP [3]. Pol μ also has a significant nucleotidyltransferase activity in the presence of Mn++ [57•, 62•, 63•]. Interestingly, TdT nucleotide binding site can accommodate both deoxyribo-nucleotide and ribo-nucleotide triphosphates (dNTPs and rNTPs). TdT shares this property with pol μ, but not with pol λ and pol β (Figure 1a). Indeed, the presence of a YF sequence motif, the so-called steric-gate at the vicinity of the 2′ OH of the nucleotide, prevents the binding of rNTPs in pol λ and pol β, whereas a GW sequence motif in TdT and pol μ (Figure 1b) increases the size of the nucleotide binding pocket, allowing binding of both dNTPs and rNTPs [64, 65]. However, addition of rNTPs by TdT stops after few incorporations on ssDNA [66, 67]. This can be interpreted by noting that the path of the primer is constrained in a B-DNA form by the protein, especially through a Na+ ion coordinated by the HhH2 motif (Figure 1b) at the level of the penultimate phosphate of the primer, and this B-DNA form is not suitable for an RNA backbone. TdT can also incorporate efficiently various un-natural bases [68, 69]. An interesting consequence of the large tolerance of TdT on the incoming nucleotide is to use it for making polymers of un-natural DNA, using nucleotides modified either in the sugar moiety or the base moiety [70] or for click chemistry [71]. Also a recent application for FISH experiments and the design of RNA capture probes can be found in [72].