Abstract
To investigate protein-DNA interactions, we have synthesized a versatile phthalimide-protected 5-(3-aminopropyl)-2'-deoxyuridine nucleoside probe. The modified residue was incorporated into deoxyoligonucleotides by automated synthesis. The standard oligonucleotide workup also exposed the pendent amino group, which was found to react with either fluorescent labelling agents or, as detailed below, a photoactivatable cross-linking agent. In the dark, a strand with a photolabile group adjacent to the 3' end served as a primer for synthetic template-directed DNA synthesis by the Klenow fragment of E. coli DNA polymerase I, by bacteriophage T4 DNA polymerase, and by avian myeloblastosis virus (AMV) reverse transcriptase. Brief illumination with 302 nm light afforded covalent complexes between DNA and the polymerases; labelling of AMV reverse transcriptase was predominantly in the beta subunit.
Full text
PDF












Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barone A. D., Tang J. Y., Caruthers M. H. In situ activation of bis-dialkylaminophosphines--a new method for synthesizing deoxyoligonucleotides on polymer supports. Nucleic Acids Res. 1984 May 25;12(10):4051–4061. doi: 10.1093/nar/12.10.4051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Basu A., Modak M. J. Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I. Biochemistry. 1987 Mar 24;26(6):1704–1709. doi: 10.1021/bi00380a032. [DOI] [PubMed] [Google Scholar]
- Bryant F. R., Johnson K. A., Benkovic S. J. Elementary steps in the DNA polymerase I reaction pathway. Biochemistry. 1983 Jul 19;22(15):3537–3546. doi: 10.1021/bi00284a001. [DOI] [PubMed] [Google Scholar]
- Dreyer G. B., Dervan P. B. Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II). Proc Natl Acad Sci U S A. 1985 Feb;82(4):968–972. doi: 10.1073/pnas.82.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dörper T., Winnacker E. L. Improvements in the phosphoramidite procedure for the synthesis of oligodeoxyribonucleotides. Nucleic Acids Res. 1983 May 11;11(9):2575–2584. doi: 10.1093/nar/11.9.2575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Evans R. K., Haley B. E. Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. Biochemistry. 1987 Jan 13;26(1):269–276. doi: 10.1021/bi00375a037. [DOI] [PubMed] [Google Scholar]
- Evans R. K., Johnson J. D., Haley B. E. 5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5382–5386. doi: 10.1073/pnas.83.15.5382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lehman I. R. T4 DNA polymerase. Methods Enzymol. 1974;29:46–53. [PubMed] [Google Scholar]
- Lewis R. V., Roberts M. F., Dennis E. A., Allison W. S. Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2. Biochemistry. 1977 Dec 13;16(25):5650–5654. doi: 10.1021/bi00644a041. [DOI] [PubMed] [Google Scholar]
- Mizrahi V., Benkovic P., Benkovic S. J. Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5769–5773. doi: 10.1073/pnas.83.16.5769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ollis D. L., Brick P., Hamlin R., Xuong N. G., Steitz T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 1985 Feb 28-Mar 6Nature. 313(6005):762–766. doi: 10.1038/313762a0. [DOI] [PubMed] [Google Scholar]
- Shimkus M., Levy J., Herman T. A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns. Proc Natl Acad Sci U S A. 1985 May;82(9):2593–2597. doi: 10.1073/pnas.82.9.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolfes H., Fliess A., Winkler F., Pingoud A. Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases. Eur J Biochem. 1986 Sep 1;159(2):267–273. doi: 10.1111/j.1432-1033.1986.tb09863.x. [DOI] [PubMed] [Google Scholar]