The operon for carbamoyl phosphate synthetase (CarAB) in the thymidine biosynthesis regulatory pathway was derepressed by disrupting three known repressors (purR, pepA and argR). Combinatorial disruption of three repressors increased CarA expression levels in accordance with degree of disruption, which had a positive correlation with thymidine production. By simultaneous disruption of three repressors (BLdtugRPA), CarA expression level was increased by 3-fold compared to the parental strain, leading to an increased thymidine yield from 0.25 to 1.1 g thymidine l−1. From BLdtugRPA, we established BLdtugRPA24 by transforming two plasmids expressing enzymes in the thymidine biosynthetic pathway and obtained 5.2 g thymidine l−1 by Ph-stat fed-batch fermentation.
As an example of the factors involved, consider the fluorinated pyrimidines, 5-fluorouracil (FUra) and 5-fluorodeoxyuridine (FdUrd), analogues used for four decades in treating various cancers. It was established in 1958 (9) that these drugs are converted in vivo to 5-fluorodeoxyuridine monophosphate (FdUMP), an analogue of deoxyuridine monophosphate, the substrate for thymidylate synthase (Fig. 1; see Deoxyribonucleotide Biosynthesis And Degradation), and that FdUMP is a potent inhibitor of thymidylate synthase and, hence of DNA replication. Figure 1 shows also the metabolic pathways that both activate these analogues and divert them from their desired endpoint (10). From the figure, one can see that coadministration with FdUrd of a thymidine phosphorylase inhibitor should increase the effectiveness of the analogue by blocking its catabolism. Note that there are multiple routes for activation of FUra; note also that FUra can enter pools of RNA precursors which, in principle, could limit its selectivity by diminishing the specificity of its effect against DNA synthesis. There is evidence, however, that, in some tumors, the effectiveness of FUra actually depends in part on its incorporation into RNA, where it stimulates translational miscoding.
Synthesis and Evaluation of α-Thymidine Analogues as …
Lee M, Wang L and Chang Z (2014) The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress. Nucleic Acids Research 42: 4972–4984.
THYMIDINE (PYRIMIDINE NUCLEOSIDE)
Thymidine kinase, thymidilate kinase and thymidilate synthetase activities have been studied during differentation in culture of muscle cells from chick embryo both in dplicating myoblasts and in post-mitotic myotubes. Thymidine kinase activity starts decreasing long before fusion, when myoblasts are still actively engaged in DNA synthesis and it reaches a minimum value at fusion (approx. 70 h of culture). Also thymidilate synthetase undergoes a precocious alteration of its activity which is low at 24 h culture, it increases up to 48 h and drops to a very low level after fusion. Finally, thymidilate kinase activity remains constant during the duplicative period and declines only after fusion. Pulse-labelling experiments with [3H]thymidine and [3H]deoxycytidine show that the incorporation of these precursors into the deoxynucleotide pool and into DNA parallels the patterns of the enzymatic activities measured in vitro.
Thymidine | Article about Thymidine by The Free Dictionary
Woeller CF, Anderson DD, Szebenyi DM, et al. (2007) Evidence for small ubiquitin‐like modifier‐dependent nuclear import of the thymidylate biosynthesis pathway. Journal of Biological Chemistry 282: 17623–17631.
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MacFarlane AJ, Anderson DD, Flodby P, et al. (2011) Nuclear localization of the thymidylate synthesis pathway is required to prevent uracil accumulation in DNA. Journal of Biological Chemistry 286: 44015–44022.