Replacement of Thymidine by a Modified Base in the E. coli ge-nome (original) (raw)
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Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome
Journal of the American Chemical Society, 2016
Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.
Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity
Journal of bacteriology, 1985
To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T-* G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ungstrain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis. Incubation of bacterial thymine auxotrophs in the absence of thymine, under conditions otherwise conducive to growth, has been found to provoke a number of thymineless phenomena including mutagenesis (for a review, see reference 19). The results of several studies led to the conclusion that thymine starvation causes primarily A:T-* G:C transitions,
Biochemistry, 1998
Various forms of oxidative stress, including γ-radiolysis and UV irradiation, result in the formation of damaged bases. (5R)-Thymidine C5-hydrate is one of several modified nucleosides produced from thymidine under these conditions. N-(2-Deoxy--D-erythro-pentofuranosyl)-N-3-[(2R)-hydroxyisobutyric acid]urea or RRT is the respective fragmentation product formed from (5R)-thymidine C5hydrate upon hydrolysis. This modified nucleoside has potential mutagenic or lethal properties. No enzymatic activity responsible for the removal of RRT has been identified. We report here that when present in DNA, RRT is a substrate for two purified enzymes from Escherichia coli involved in the repair of oxidized bases: the Nth and the Fpg proteins. The Fpg protein removes the RRT lesion more efficiently than the Nth protein. This is the first example of efficient excision of a ring-opened form of a pyrimidine by the Fpg protein. The high efficacy of the Fpg protein suggests that it is likely to be involved in vivo in the excision of RRT. The kinetics of the reaction of the Fpg protein with DNA containing RRT suggest substrate inhibition. Duplex oligodeoxynucleotides containing RRT positioned opposite T, dG, dC, and dA were cleaved efficiently by both enzymes, although the profiles of activity of the two enzymes were different. The Nth enzyme preferentially excises RRT when opposite a dG, followed by RRT‚dA, RRT‚T, and RRT‚dC. For the Fpg protein, the order is RRT‚dC g RRT‚dG ∼ RRT‚T > RRT‚dA. Moreover, we show that human cell extract exhibits an activity that excises RRT from an oligonucleotide, suggesting that human homologues of the Nth and/or Fpg proteins could be involved in repair of this lesion in human cells.
Proceedings of the National Academy of Sciences of the United States of America, 2018
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the phage ΦW-14 and the phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the phage M6 and the phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-emdU) and 5-(2-aminoethyl)uridine (5-edU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is...
Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity
Proceedings of the National Academy of Sciences, 1992
The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn'", which was changed to aspartic acid. The side-chain amide of Asn'77 forms hydrogen bonds with 04 and N3 of dUMP bound to the crystalline enzyme []. This Asn is invariant in all natural sequences for TS known. The values of kc.t for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kn. of wild-type TS with dUMP as substrate. TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-
Synthesis and enzymatic incorporation of a novel, bicyclic pyrimidine nucleoside: a thymidine mimic
Tetrahedron Letters, 2003
Nucleophilic ring-opening and rearrangement reaction of a furanopyrimidine nucleoside with anhydrous hydrazine provided a novel, 6,6-bicyclic pyrimidopyridazin-7-one nucleoside (dH, 4), whose structure was confirmed by X-ray crystallography. This novel nucleoside was converted to its 5%-triphosphate (dHTP) for studies with DNA polymerases and incorporated into a template by using standard phosphoramidite chemistry. In the template, dH directed the incorporation of dATP and to a lesser extent dGTP into the transcript and dHTP was efficiently incorporated at the 3%-end of a primer opposite dA using both exonuclease free Klenow fragment (KF exo-) and Taq DNA polymerases and extended with natural dNTPs.
Archives of Biochemistry and Biophysics, 1978
Through a new approach, we have sought t o isolate ultraviolet light (UV)sensitive and DNA repair mutant Chinese hamster fibroblasts. T h e procedure consisted of 1) mutation induction by 5-bromodeoxyuridine (BrdU)-blacklight and UV treatments; 2) incorporation of H-thymidine in reoair-Droficien! cells at high temperature (38.5"C) following UV damage; 3) cold holding (4.0 C) of these cells to induce tritium killing; and 4) recovery and testing of repairdeficient and UV-sensioive cells which have survived and formed colonies at low temperature (34.0 C). In our initial a t t e m p t a t this protocol, we isolated 72 surviving colonies f r o m 2 x 10' cells plated f o r selection. Of t h e 7 2 colonies, 20 demonstrated potential interest and four were selected f o r extensive study. O n e , identified as UVS-7, is slightly more sensitive to UV, but n o t sensitive to X rays o r N-acetoxy-2-acetylaminofluorene (NAc-AAF). T h e m u t a n t exhibits a highly reduced level of unscheduled D N A synthesis (UDS), as compared t o the parental line. T w o additional lines, UVS-40 and UVS-44, are sensitive t o UV, X ray, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and NAc-AAF, but exhibit normal UDS. A fourth line, UVr-23, has enhanced UDS, is resistant t o UV, b u t exhibits n o difference in sensitivity t o X ray or NAc-AAF. These mutants are all stable, and should b e useful for the study of mammalian DNA repair processes and mechanisms of mutagenesis.
Journal of Molecular Biology, 1974
The requirement for pseudouridine and other uridine-derived minor nucleotides for activity of transfer RNA in several of the intermediate steps in protein synthesis wae examined using a purified preparation of Escherichia wli dine transfer RNA in which the uridine and uridine-derived nucleotides were replaced by S-fluorouridine. The degree of substitution was 87% or better for uridine, pseudouridine, ribothymidine, dihydrouridine, and 4-thiouridine, and s.t least 75% for uridine-5-oxyacetic acid. Each of these nucleotides, except for uridine, occurs only once in this transfer RNA species. The rate and yield of ternary complex formation with elongation factor Tu-GTP of E. wli, the rate and extent of elongation factor-dependent binding to ribosomes at 10 mM-Mga + , and the rate and extent of synthesis of the co-polypeptide (Phe,,Val) dependent on poly(U,,G) were all unchanged when the fluorouridine-conttiing transfer RNA was used in place of the normal control. In all yield sssays, the amount of product formed was proportional to the amount of valyl-tRNA added. Non-enzymatio binding to ribosomes in the presence of tetracycline was more efficient for the fluorouridine-substituted tRNA than for the control. At 16 to 20 mM-Mg a + the polynucleotide-dependent binding, as & percentage of tRNA added, was 44% for the control and 05% for the modified tRNA, while at 5 mM-Mga+, the figures were 10% and 40%, respectively. We conclude from these results that there is no essential requirement for pseudouridine or ribothymidine in the GT+C loop of tRNA for its proper functioning in protein synthesis in vitro. ConfIrming earlier work, dihydrouridine and 4thiouridine are also not essential.