5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA (original) (raw)
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A Binary System of Photoreagents for High-Efficiency Labeling of DNA Polymerases
Biochemical and Biophysical Research Communications, 2001
To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.
2001
To introduce photoreactive dNMP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[ N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3aminopropenyl-1]-and 5-{ N-[ N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-trans-3aminopropenyl-1}-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase β-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at a predetermined position of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase β and human replication protein A (hRPA) were carried out. It was shown that DNA polymerase β and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.
Russian Journal of Bioorganic Chemistry, 2000
Abstraet--A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3amino-l-propen-l-yl]-2',3'-dideoxyuridine 5'-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2'-deoxyuridine 5'-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTrP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase 1~ and to terminate DNA synthesis. A 5'-32p-labeled primer with a photoreactive group at the 3'-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.
Highly Efficient Labeling of DNA Polymerases by a Binary System of Photoaffinity Reagents
2002
Thermus thermophilus B35; FAB 4 dUTP) 5 [N (2,3,5,6 tetra fluoro 4 azidobenzoyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; FAB 4 ddUTP) 5 [N (2,3,5,6 tetrafluoro 4 azidobenzoyl) 3 amino trans propenyl 1] 2′,3′ dideoxyuridine 5′ triphosphate; FAB 9 dUTP) 5 [N [[(2,3,5, 6 tetrafluoro 4 azidobenzoyl) butanoyl] amino] 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; Pyr 8 dUTP) 5 [N (4 (1 pyrenyl) butylcarbonyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; Pyr 6 dUTP) 5 [N (4 (1 pyrenyl) ethylcarbonyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate.
Proceedings of the National Academy of Sciences, 1986
Replication of UV-irradiated circular singlestranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase HI holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage 4X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase m holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
FEBS Letters, 1999
To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase L L (pol L L). 5P-[ QP P]-labeled primer was elongated in situ by pol L L with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365^450 nm), can activate the photoreagent, crosslinking it to pol L L as a result of fluorescence resonance energy transfer. The initial rate of pol L L photomodification was shown to increase by a factor of ten. The selectivity of pol L L photosensitized modification was proved by adding human replication protein A.
We report the preparation and use of an N-methyl picolinium carbamate protecting group for applications in a phototriggered nonenzymatic DNA phosphoramidate ligation reaction. Selective 5′-amino protection of a modified 13-mer oligonucleotide is achieved in aqueous solution by reaction with an N-methyl-4-picolinium carbonyl imidazole triflate protecting group precursor. Deprotection is carried out by photoinduced electron transfer from Ru(bpy) 3 2+ using visible light photolysis and ascorbic acid as a sacrificial electron donor. Phototriggered 5′-amino oligonucleotide deprotection is used to initiate a nonenzymatic ligation of the 13-mer to an imidazole activated 3′-phospho-hairpin template to generate a ligated product with a phosphoramidate linkage. We demonstrate that this methodology offers a simple way to exert control over reaction initiation and rates in nonenzymatic DNA ligation for potential applications in the study of model protocellular systems and prebiotic nucleic acid synthesis.
Characterization of (6-4) photoproduct DNA photolyase
The Journal of biological chemistry, 1994
Pyrimidine (6-4) pyrimidone photoproduct is the second most abundant UV photoproduct in DNA. Recently, it was reported that Drosophila melanogaster cell-free extracts restored the biological activity of (6-4) photo-product-containing DNA in a light-dependent reaction (Todo, T., Takemori, H., Ryo, H., Ihara, M., Matsunaga, T., Nikaido, O., Sato, K., and Nomura, T. (1993) Nature 361, 372-374) concomitant with the loss of (6-4) photoproduct antigenic sites and (6-4) photoproduct-caused alkali-labile sites. In the present study we show that the (6-4) photoproduct but not its Dewar isomer is the substrate for the enzyme, that the enzyme has an action spectrum peak at 400 nm, and that the efficiency of repair per incident photon is very low compared with cyclobutane pyrimidine dimer photolyases. Furthermore, we provide evidence that the (6-4) photoproduct photolyase converts the photoproduct to unmodified bases probably through an oxetane intermediate.