Synthesis and Characterization of (d)NTP Derivatives Substituted with Residues of Different Photoreagents

Abstract

Chemical cross-linking agents having a photoactivable azido group are promising for the study of the spatial organization of biopolymers. We describe here a variety of (d)NTPs derivatives (6a, 6b, 7, 11, 12, 14, and 16) bearing the residues of three different photoreagents containing an aromatic azido group (1a, 2a, and 3a). These conjugates provide a wide choice of instruments to investigate nucleic acid-nucleic acid and nucleic acid-protein interaction. The synthesis of new photoreagent 2a has been also fulfilled. This compound is the most attractive for affinity modification of the nucleic acids.

Analyzing the Handoff of DNA from UvrA to UvrB Utilizing DNA-Protein Photoaffinity Labeling

“…3E, (10, 15). As stated above, we have strategically designed our substrates to contain a cross-linking probe on either the damaged or non-damaged strand (XL1 26 and XL2 25 C, a photoreactive AZ functional group was coupled to the backbone of a phosphorothioate-containing (at the phosphodiester linkage, S-1) oligonucleotide. The terminal azido group (N 3 ) in each reagent is activated by exposure to UV light (365 nm) allowing DNA-protein cross-linking to occur via a highly reactive nitrene intermediate.…”

“…5-{[N-(4-azidotetrafluorobenzylideneaminooxy)methylcarbamoyl]-trans-3-aminopropenyl-1}-2Ј-deoxyuridine-5Ј-triphosphate (XL1, Fig. 2A) was synthesized and characterized as described previously (25). Synthesis of exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2Ј-deoxycytidine-5Ј-triphosphate (XL2, Fig.…”

“…Gapped Heteroduplex Substrates, Gap 25 and Gap 26 -To generate gapped heteroduplex substrates, both a 25-mer oligonucleotide (5Ј-GACTACGTACTGTTACGGCTCCATC-3Ј) and a 24-mer oligonucleotide (5Ј-GACTACGTACTGTTACGGCTCCAT-3Ј) were 5Ј-end-labeled with OptiKinase (United States Biochemical Corp.) and [␥- 32 P]ATP (3000 Ci/mmol, Amersham Biosciences) as described above. The reaction was terminated by the addition of 20 mM EDTA, and the enzyme was heat-denatured by incubation for 10 min at 65°C.…”

“…Nicked Heteroduplex Substrates, dT 26 , dC 25 , XL1 26 , and XL2 25 -Nicked heteroduplex substrates were prepared by incubating the gapped heteroduplex substrates Gap 26 and Gap 25 (ϳ22 pmol) with 100 pmol of dTTP and XL1, or 400 pmol dCTP and XL2, respectively, in the presence of human pol ␤ (45 pmol, a generous gift from Drs. Rajendra Prasad and Samuel Wilson, NIEHS, National Institutes of Health) in 13 l of 1ϫ pol ␤ buffer (50 mM Tris-HCl (pH 7.5), 50 mM KCl, 20% glycerol) for 1 h at 37°C.…”

“…DNA-UvrAB Cross-linking Assay-The 5Ј-end-labeled duplex DNA containing the cross-linker moiety (2 nM, XL1 26 , XL2 25 , or S-1 AZ ) was incubated with the UvrAB proteins (200 nM B. caldotenax UvrA, 1000 nM B. caldotenax wild-type (WT) UvrB or mutants) in 20 l of reaction volume containing UvrAB cross-linking buffer (50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl 2 , 1 mM ATP) at 55°C for up to 30 min in the dark. Reaction vials were immediately transferred to a platform 5 cm below a 365 nm UV lamp (UVP, Blak-Ray Longwave UV Mercury lamp, 100 watts) for indicated time points, typically 5 min).…”