| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892
Correspondence: Address reprint requests and inquiries to Nicolas Sluis-Cremer, Tel.: 412-648-8457; Fax: 412-648-8521; E-mail: cremern{at}dom.pitt.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT ACKNOWLEDGEMENTSREFERENCES |
|---|
The effects of ionizing radiation on DNA have been widely studied for many years (1
), although radiation target analyses had only been reported before well-defined preparations and advanced techniques became available. Recently it was demonstrated that radiation damage in RNA ribozymes occurred only locally in the molecule (2), which is in sharp contrast with the well-established spread of radiation damage throughout polypeptides (3,4). This surprising result stimulated questions about the nature of radiation damage in other nucleic acid complexes, in particular DNA. In this study, radiation analyses of defined DNA template/primer (T/P) complexes were conducted in the absence and presence of HIV-1 reverse transcriptase (RT), an enzyme that binds these nucleic acid complexes with high affinity and previously has been studied by radiation target analysis (5).
Two different DNA T/P complexes were subjected to radiation inactivation analyses. One was a homopolymeric poly(C)30-poly(G)18 T/P (14.4 kDa) that consisted of an 18-nucleotide homopolymeric primer (dG18; 5865 Da) annealed to a 30-nucleotide homopolymeric template (dC30; 8615 Da). The other was a heteropolymeric T/P (designated (T)30-(P)18; 15.8 kDa) that consisted of an 18-nucleotide heteropolymeric primer (5'-CCTGTTCGGGCGCCACTG-3'; 5756.6 Da) annealed to a complementary 30-nucleotide heteropolymeric template (5'-GAAAATCTCTAGCAGTGGCGCCCGAACAGG-3'; 10089.2 Da). All DNA oligomers were synthesized and HPLC-purified by Integrated DNA Technologies (Coralville, IA). DNA T/P complexes were formed by combining equimolar concentrations of template and primer at 90°C and allowing the mixture to slowly cool to ambient room temperature. Samples for irradiation were diluted to 2 mg/ml in 20 mM Tris-HCl (pH 7.5, 20°C) and 100 mM NaCl. This concentration of macromolecule has previously been shown to be high enough to yield meaningful radiation target sizes and avert the need for free radical scavengers (6). Radiation exposures of 0–100 Mrads were obtained as described (7) from 10 MeV electrons produced by linear accelerators at the Armed Forces Radiobiology Research Institute or at the National Institute of Standards and Technology. Following irradiation, the surviving double-stranded DNA species in the poly(C)30-poly(G)18 samples were quantified using the Quant-iT PicoGreen double-stranded DNA quantitation assay (Molecular Probes, Invitrogen, Carlsbad, CA). This assay can selectively detect as little as 25 pg/mL of double-stranded DNA in the presence of single-stranded DNA, RNA, and free nucleotides and is linear over three orders of magnitude with little sequence dependence. The template and primer strands of the (T)30-(P)18 heteropolymeric T/P complex were analyzed independently using denaturing polyacrylamide gel electrophoresis to separate the strands, and SYBR-Gold reagent (Molecular Probes, Invitrogen) to detect the oligonucleotides. The radiation inactivation profiles of both DNA T/P complexes were identical, whether measured as total double-stranded DNA or individually as the template and primer oligonucleotides (Fig. 1). In all cases the observed target sizes were 
16,000 Da (Table 1), which corresponds to their double-stranded mass. Theoretical inactivation curves for a 5-kDa and a 10-kDa target clearly do not fit the data (Fig. 1).
|
|
). The radiation target sizes of HIV-1 are also unaffected by the presence of DNA T/P complexes (5). In this study, recombinant HIV-1 RT was purified according to published techniques (8,9) and the target sizes of the enzyme were determined by SDS-gel electrophoresis and by analysis of the residual RDDP activity, as described previously (5). The radiation inactivation profiles of HIV-1 RT determined by SDS-PAGE and RDDP activity in this study (Table 2 and Fig. 2) correlate with the heterodimeric structure of the enzyme and are essentially identical to those determined previously (5). Therefore this enzyme acted as an internal control for the new study. As described above, the irradiated samples were also analyzed for surviving DNA T/P complexes. The observed target sizes for both T/P complexes did not appear to be influenced by their interactions with HIV-1 RT (Table 2).
|
|
16 kDa, with very small error. Thus, a primary ionization in any single chain of DNA results ultimately in cleavage of another polynucleotide chain to which it is basepaired.
As described previously (5), the radiation target sizes of HIV-1 RT were unaffected by the presence of DNA T/P complexes, indicating the absence of any transfer to the protein of radiation energy deposited in the DNA. These results clearly demonstrate the lack of energy transfer in the opposite direction. The crystal structure of an RT-T/P complex (10) shows that the principal contacts between the RT protein and T/P are via the deoxyribose sugar ring. Previous radiation studies of oligosaccharides (11), RNA ribozymes (2), and large DNA plasmids (T. J. Anchordoquy, M. d. C. Molina, and E. S. Kempner, unpublished data) suggested that the transfer of radiation-deposited energy was prevented by the presence of sugar rings in the polymer. This phenomenon could explain the isolation of radiation damage to RT from T/P and vice versa. In this case, the blockage of energy transfer from a polynucleotide may be due to the deoxyribose that is noncovalently bound to a polypeptide. A brief report of electron spin resonance signals in irradiated chromatin (13) indicated electron transfer, but not electron hole transfer, from histone into DNA. The association between histone and DNA is principally through noncovalent interactions with DNA phosphate groups (14). The conflicting results from irradiated RT-T/P and chromatin could be explained by the difference in sugar ring interactions.
The T/P inactivation curve appears to be a simple exponential, indicating only a single-sized target. Therefore the efficiency of energy transfer between DNA strands must approach 100%. If there were subsets of T/P complexes that did not permit energy transfer, the inactivation curve would not be a simple exponential but rather the sum of the curves from the individual subsets (15). This theoretical model does not fit the data at all.
Previous studies of radiation effects on DNA and of energy transfer between strands (1) were principally assayed by "double-strand breaks". These are cleavages of both polymer backbones where the breaks are within a very few basepairs of each other. These differ from polymer scissions detected in this study in which there is no restriction as to the relative positions of the two breaks. The principal conclusions of this study, therefore, is that in these particular T/P DNA complexes, when exposed in the frozen state to ionizing radiation, a single radiation interaction in either strand results in scission of that strand as well as a scission somewhere in the opposite strand. No damage is detected in either the protein RT or the T/P when the other component is damaged.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
|---|
This work was partially supported by National Institutes of Health grant NIH R01 GM068406 to N.S.-C. and by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health to E.S.K.
Submitted on February 13, 2006; accepted for publication February 24, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
|---|
2. Bernstein, S. L., and E. Kempner. 1996. Radiation target analysis of RNA. Proc. Natl. Acad. Sci. USA. 93:6410–6414.
3. Kempner, E. S., K. W. Cole, and F. H. Gaertner. 1982. Functional unit of the arom conjugate in Neurospora. J. Biol. Chem. 257:8919–8921.
4. Kempner, E. S. 2001. Effects of high-energy electrons and gamma rays directly on protein molecules. J. Pharm. Sci. 90:1637–1646.[CrossRef][Medline]
5. Sluis-Cremer, N., E. Kempner, and M. A. Parniak. 2003. Structure-activity relationships in HIV-1 reverse transcriptase revealed by radiation target analysis. Protein Sci. 12:2081–2086.
6. Kempner, E. S., and J. H. Miller. 1990. Direct effects of radiation on the avidin-biotin system. J. Biol. Chem. 265:15776–15781.
7. Harmon, J. T., T. B. Nielsen, and E. S. Kempner. 1985. Molecular weight determinations from radiation inactivation. Methods Enzymol. 117:65–94.[Medline]
8. Le Grice, S. F., C. E. Cameron, and S. J. Benkovic. 1995. Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Methods Enzymol. 262:130–144.[Medline]
9. Le Grice, S. F., and F. Gruninger-Leitch. 1990. 1990. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur. J. Biochem. 187:307–314.[Medline]
10. Ding, J., K. Das, Y. Hsiou, S. G. Sarafianos, A. D. Clark Jr., A. Jacobo-Molina, C. Tantillo, S. H. Hughes, and E. Arnold. 1998. Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 A resolution. J. Mol. Biol. 284:1095–1111.[CrossRef][Medline]
11. von Sonntag, C. 1980. Free-radical reactions of carbohydrates as studied by radiation techniques. Adv. Carbohydr. Chem. Biochem. 37:7–77.
12. Reference deleted in proof.
13. Cullis, P. M., G. D. D. Jones, M. C. R. Symons, and J. S. Lea. 1987. Electron transfer from protein to DNA in irradiated chromatin. Nature. 330:773–774.[CrossRef][Medline]
14. Davey, C. A., D. F. Sargent, K. Luger, A. W. Maeder, and T. J. Richmond. 2002. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 A resolution. J. Mol. Biol. 319:1097–1113.[CrossRef][Medline]
15. Kempner, E. S. 1995. The mathematics of radiation target analyses. Bull. Math. Biol. 57:883–898.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
