br Methods for assessing TLS

Methods for assessing TLS While the precise quantification of restricted DNA synthesis events is possible (e.g. unscheduled DNA synthesis (UDS) reveals NER), so far, it is impossible to identify TLS stretches of only a few nucleotides within the background of bulk DNA replication of normal DNA. Nevertheless, TLS efficiency may be inferred indirectly by monitoring various accepted TLS markers (Fig. 2).
Negative regulators of TLS
Concluding remarks and perspectives While some aspects of the regulation of TLS by USP1, p21 and Spartan have been revealed, a number of issues require immediate attention. While it is accepted that the consequences of the inactivation of a single Y-pol must be different from those arising from the global block of all Y-pols, with the exception of p21 [45], the analysis of most inhibitors has been restricted to Polη [36], [51], [53], [54], [55], [56], [57], [60], [61]. Moreover, the overexpression/stabilization of TLS inhibitors should be exploited to support their negative role in TLS. In fact, the extensive use of gain-of-function-tools combined with the analysis of all Y-family pols served to define p21 as a global negative regulator of TLS in UV damage [45], while similar experiments with USP1 and Spartan are yet to be performed. Application of the DNA fiber assay has shown that the functions of the TLS inhibitors do not totally overlap. After UV-irradiation, p21 degradation increases DNA elongation, thus supporting its role as a global TLS inhibitor [45], while Spartan dysfunction causes the opposite effect [56], [57]. Intriguingly, the role of USP1 in DNA Nitazoxanide after UV irradiation has not been yet reported. Moreover, loss of either negative or positive TLS regulators cause hypersensitivity to DNA damage, which might indicate that an “appropriate” level of TLS events is required for cell viability, e.g. [53]. Another important issue that requires clarification is the contribution of TLS regulators to replication of undamaged DNA. TLS pols are certainly required for the synthesis across difficult-to-replicate DNA structures such as common fragile sites [4], but their participation in undamaged DNA replication must be restricted to minimize mutagenesis and other genomic instability parameters [39]. While USP1 has a well-documented role in the protection of undamaged DNA replication [39], diminished levels of Spartan during unperturbed replication affect the TLS parameter of DNA elongation [56]. This emphasizes the need for research to explore the contribution of TLS inhibition to the successful execution of the replication program in the absence of stress. The information discussed in this review indicates that USP1 may have a more prominent role in the prevention of unleashed Y-pol loading on undamaged DNA than on the onset of TLS. On the other hand, p21 has been placed directly at the on-switch for TLS [42] and more conflicting evidence places Spartan at the off-switch for TLS [51], [54], [57] (Fig. 3). In this regard, it is important to mention that recent reports bring the PCNA-interacting protein PAF15 and the ubiquitin-like protein ISG15 into play, being both factors potentially involved in the restoration of replicative DNA synthesis after TLS finalization [60], [61]. PAF15 may also prevent unleashed loading of Polη to undamaged DNA [60]. Additionally, emerging evidence highlights potential cross-regulation between TLS inhibitors, as USP1 and Spartan have been functionally linked [55]. Understanding the interconnections between TLS-regulators should foster the comprehension of the mechanisms that limit mutagenesis to optimal levels in cells.
Acknowledgements
Introduction Terminal deoxynucleotidyltransferase (TdT) was one of the first eukaryotic DNA polymerases purified in the early 1960s [1], from calf thymus extracts. However, instead of the expected classical templated polymerase activity, the biochemical characterization of TdT revealed an efficient untemplated polymerase (nucleotidyltransferase) activity [2, 3], especially in the presence of divalent transition metal ions [4]. In vivo, the function of TdT was only fully understood in the eighties [5, 6, 7], after the discovery of V(D)J recombination [8, 9, 10]. During this process, TdT adds random nucleotides (N-segments) at the V-D and D-J junctions in heavy chains of immunoglobulins (Ig) and T-cell receptors, thereby contributing significantly to the diversity of the immune repertoire [11, 12]. Subsequently, it was revealed that the V(D)J uses the same machinery [13, 14] as the one of Non-Homologous End Joining (NHEJ) that repairs DNA double-strand breaks (DSB). This machinery includes a recognition complex (Ku heterodimer, DNA-PKcs), DNA end-processing enzymes such as a nuclease (Artemis or Metnase) and a DNA polymerase (pol X), as well as a ligation complex (Lig IV, XRCC4, XLF) [15]. The DNA polymerase is a member of the family polX that includes not only TdT, but also pol λ [] and pol μ [17•, 18•], the last two participating to both NHEJ and V(D)J recombination [19, 20, 21, 22, 23]. All three polymerase domains X-ray structures have been determined to high resolution [24•, 25•, 26•] but the only one that was crystallized in a DNA-bridging context is TdT [27••, 28••]. Here we focus on TdT and on the biological implications of these new structures.