Abstract
We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II) cleaves double helical DNA with low sequence specificity. Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs. We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.
Full text
PDF![5470](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5dbd/346925/beff284f8f8e/pnas00457-0048.png)
![5471](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5dbd/346925/eaa0b42686da/pnas00457-0049.png)
![5472](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5dbd/346925/85b2b3ff3f7c/pnas00457-0050.png)
![5473](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5dbd/346925/87a71552b14b/pnas00457-0051.png)
![5474](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5dbd/346925/2be3cff7044f/pnas00457-0052.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Berlin V., Haseltine W. A. Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J Biol Chem. 1981 May 25;256(10):4747–4756. [PubMed] [Google Scholar]
- Berman H. M., Neidle S., Zimmer C., Thrum H. Netropsin, a DNA-binding oligopeptide structural and binding studies. Biochim Biophys Acta. 1979 Jan 26;561(1):124–131. doi: 10.1016/0005-2787(79)90496-9. [DOI] [PubMed] [Google Scholar]
- Bresloff J. L., Crothers D. M. Equilibrium studies of ethidium--polynucleotide interactions. Biochemistry. 1981 Jun 9;20(12):3547–3553. doi: 10.1021/bi00515a038. [DOI] [PubMed] [Google Scholar]
- Burger R. M., Peisach J., Horwitz S. B. Mechanism of bleomycin action: in vitro studies. Life Sci. 1981 Feb 16;28(7):715–727. doi: 10.1016/0024-3205(81)90153-3. [DOI] [PubMed] [Google Scholar]
- D'Andrea A. D., Haseltine W. A. Sequence specific cleavage of DNA by the antitumor antibiotics neocarzinostatin and bleomycin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3608–3612. doi: 10.1073/pnas.75.8.3608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- GOLDBERG I. H., RABINOWITZ M., REICH E. Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2094–2101. doi: 10.1073/pnas.48.12.2094. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kastrup R. V., Young M. A., Krugh T. R. Ethidium bromide complexes with self-complementary deoxytetranucleotides. Demonstration and discussion of sequence preferences in the intercalative binding of ethidium bromide. Biochemistry. 1978 Nov 14;17(23):4855–4865. doi: 10.1021/bi00616a002. [DOI] [PubMed] [Google Scholar]
- Krylov A. S., Grokhovsky S. L., Zasedatelev A. S., Zhuze A. L., Gursky G. V., Gottikh B. P. Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs. Nucleic Acids Res. 1979 Jan;6(1):289–304. doi: 10.1093/nar/6.1.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LePecq J. B., Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol. 1967 Jul 14;27(1):87–106. doi: 10.1016/0022-2836(67)90353-1. [DOI] [PubMed] [Google Scholar]
- Luck G., Triebel H., Waring M., Zimmer C. Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers. Nucleic Acids Res. 1974 Mar;1(3):503–530. doi: 10.1093/nar/1.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Müller W., Crothers D. M. Interactions of heteroaromatic compounds with nucleic acids. 1. The influence of heteroatoms and polarizability on the base specificity of intercalating ligands. Eur J Biochem. 1975 May;54(1):267–277. doi: 10.1111/j.1432-1033.1975.tb04137.x. [DOI] [PubMed] [Google Scholar]
- Müller W., Crothers D. M. Studies of the binding of actinomycin and related compounds to DNA. J Mol Biol. 1968 Jul 28;35(2):251–290. doi: 10.1016/s0022-2836(68)80024-5. [DOI] [PubMed] [Google Scholar]
- Patel D. J., Kozlowski S. A., Rice J. A., Broka C., Itakura K. Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7281–7284. doi: 10.1073/pnas.78.12.7281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Patel D. J., Kozlowski S. A., Rice J. A. Hydrogen bonding, overlap geometry, and sequence specificity in anthracycline antitumor antibiotic.DNA complexes in solution. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3333–3337. doi: 10.1073/pnas.78.6.3333. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quigley G. J., Wang A. H., Ughetto G., van der Marel G., van Boom J. H., Rich A. Molecular structure of an anticancer drug-DNA complex: daunomycin plus d(CpGpTpApCpG). Proc Natl Acad Sci U S A. 1980 Dec;77(12):7204–7208. doi: 10.1073/pnas.77.12.7204. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sobell H. M. The stereochemistry of actinomycin binding to DNA and its implications in molecular biology. Prog Nucleic Acid Res Mol Biol. 1973;13:153–190. doi: 10.1016/s0079-6603(08)60103-8. [DOI] [PubMed] [Google Scholar]
- Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
- Takeshita M., Grollman A. P., Ohtsubo E., Ohtsubo H. Interaction of bleomycin with DNA. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5983–5987. doi: 10.1073/pnas.75.12.5983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takusagawa F., Dabrow M., Neidle S., Berman H. M. The structure of a pseudo intercalated complex between actinomycin and the DNA binding sequence d(GpC). Nature. 1982 Apr 1;296(5856):466–469. doi: 10.1038/296466a0. [DOI] [PubMed] [Google Scholar]
- Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waring M. J. Complex formation between ethidium bromide and nucleic acids. J Mol Biol. 1965 Aug;13(1):269–282. doi: 10.1016/s0022-2836(65)80096-1. [DOI] [PubMed] [Google Scholar]
- Wartell R. M., Larson J. E., Wells R. D. Netropsin. A specific probe for A-T regions of duplex deoxyribonucleic acid. J Biol Chem. 1974 Nov 10;249(21):6719–6731. [PubMed] [Google Scholar]
- Wells R. D., Larson J. E. Studies on the binding of actinomycin D to DNA and DNA model polymers. J Mol Biol. 1970 Apr 28;49(2):319–342. doi: 10.1016/0022-2836(70)90248-2. [DOI] [PubMed] [Google Scholar]
- Zimmer C. Effects of the antibiotics netropsin and distamycin A on the structure and function of nucleic acids. Prog Nucleic Acid Res Mol Biol. 1975;15(0):285–318. doi: 10.1016/s0079-6603(08)60122-1. [DOI] [PubMed] [Google Scholar]