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  • br In half a century after its first biochemical


    In 2015–2016, half a century after its first biochemical characterization as a template-independent polymerase, it was shown that TdT can, firstly, assemble a DNA synapsis by itself, optimally with one micro-homology base-pair between strands [] and secondly, perform a template-dependent nucleotide incorporation across strands breaks [] in the presence of an excess of downstream dsDNA with a 3′ protruding end. Because this template-dependent activity of TdT is achieved by using an in trans template strand, instead of the usual in cis template strand, we refer to it as the in trans activity. Interestingly, this in trans templated activity was also described for pol μ [60•, 73], but without the need of an excess of the downstream DNA duplex []. In vitro biochemical experiments on chimeric constructs of TdT, involving substitution of Loop1 by pol μ’s sequence and/or reconstitution of the 5′-phosphate binding site (Figure 1), show an activity similar to pol μ, with a protein/DNA ratio of 1:1 []. The existence of templated synthesis across strand breaks has recently been described in vivo for both pol μ and pol λ [74, 75•, 76•, 77•]. The structure of TdT in complex with a DSB-DNA substrate [] is the first of its kind to be solved for a polX (Figure 2). It looks as if TdT was designed to ‘isolate’ a mini-helix made of only two ep4 pairs to stabilize and establish a fragile bridge between the upstream and downstream duplexes (Figure 1). The two upstream and downstream dsDNA are in B-DNA conformation, while the micro-homology (MH) mini-helix between them is in A-DNA conformation. L398 in Loop1 is crucial to break the helical path from the upstream dsDNA to the MH-mini helix and its role has been verified by site-directed mutagenesis [].
    Function of polX during V(D)J recombination Expression of TdT is only observed in the primary lymphoid organs, thymus and bone marrow where V(D)J recombination is active [78]. Indeed, expression of TdT is only detected during heavy chain rearrangements, but is absent from the next step where light chain rearrangements occurs [79] (Figure 4). The expression of TdT is also tightly regulated in time as it is not expressed in fetal or neo-natal life. One way this regulation is done is probably through ubiquitylation [80]. Interestingly, two TdT interacting factors (TdIF1 and TdIF2) have been identified and characterized to inhibit Tdt activity [81, 82]. Pol μ participates in light chain rearrangements during V(D)J recombination, whereas pol λ participates only in heavy chain rearrangements [] (Figure 4). It should be noted that when TdT is made to express in non-lymphoid cells, it participates in NHEJ DNA repair [84]. Also, when expressed constitutively in B-cells, it generates N-regions in both heavy and light chains [85]. It was found that inhibiting TdT in some cancer cells can kill them, such as in acute lymphoblastic leukemia cells [86]. Chemical compounds were designed and synthesized using TdT as a drug target [87, 88] and the structure of some of these compounds was solved in their bound form []. The length of the N-segments incorporated by TdT ranges from 2 to about 15–20, with two clearly different regimes in its probability distribution function: a rising phase with a peak at 4, followed by a decreasing phase [90, 91•]. Loc’h and colleagues proposed that the dual activity of TdT may correspond to these two regimes []: after the addition of a few random nucleotides on the 3′ end DNA, the downstream DNA is finally reached/sensed, at which point TdT switches to an in trans template-dependent synthesis (Figure 4). This synthesis will stop if the micro-homology base pair is of Watson-Crick type and continue otherwise, which occurs in three out of four possible cases; strikingly, this quantitatively explains the size-distribution law of the N-regions of the second phase, that exponentially decreases with a slope of −¼ [].