DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone - PubMed (original) (raw)

DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone

B Balasubramanian et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5' H > 4' H > 3' H approximately 2' H approximately 1' H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(A) Numbering scheme for the carbon atoms of deoxyribose. (B) Structures of the products of hydroxyl radical-mediated DNA cleavage. The asterisk indicates the position of the 32P radiolabel. (Left) Structure of the 3′ end of the DNA strand at the site of cleavage (indicated by the arrow). (Right) Structure of the 5′ end of the DNA strand at the site of cleavage (indicated by the arrow).

Figure 2

Figure 2

Comparison of hydroxyl radical cleavage patterns of control (all-protio) vs. deuterated DNA radiolabeled with 32P at the 5′ end. Broken line, control DNA. Solid line, deuterated DNA. Deuterated nucleotides are indicated by unfilled labels. The major cleavage product seen at each nucleotide is a strand terminated by a 3′-phosphate (see Fig. 1_B_); the minor product is the 3′-phosphoglycolate-terminated strand. Phosphoglycolate bands are marked in B by asterisks. (A) Control DNA vs. DNA containing 5′-dideuterated deoxycytidine. (B) Control DNA vs. DNA containing 4′-deuterated deoxythymidine. (C) Control DNA vs. DNA containing 3′-deuterated deoxyadenosine. (D) Control DNA vs. DNA containing 2′-dideuterated deoxyadenosine. (E) Control DNA vs. DNA containing 1′-deuterated deoxycytidine.

Figure 3

Figure 3

Comparison of hydroxyl radical cleavage patterns of control (all-protio) vs. 5′-dideuterated DNA radiolabeled with 32P at the 3′ end. Broken line, control DNA. Solid line, deuterated DNA. 5′-dideuterated deoxycytidine nucleotides are indicated by unfilled labels. The major cleavage product seen at each nucleotide is a strand terminated by a 5′-phosphate; the minor product is the 5′-aldehyde-terminated strand (see Fig. 1_B_). An aldehyde-terminated strand exhibits an unusually slow migration on the gel; previous work has established that there is a 2–3 nucleotide retardation in mobility compared with the corresponding Maxam–Gilbert product (10). Arrows show the correspondence between a 5′-phosphate-terminated product and the 5′-aldehyde-terminated product that is produced by reaction with that nucleotide.

Figure 4

Figure 4

Solvent accessibility vs. reactivity toward the hydroxyl radical. The data in Table 2 are plotted, and the best linear fit to the data is shown. Above the graph is the equation of the best-fit line, along with the correlation coefficient.

Similar articles

Cited by

References

    1. von Sonntag C. The Chemical Basis of Radiation Biology. London: Taylor & Francis; 1987.
    1. Tullius T D. Nature (London) 1988;332:663–664. - PubMed
    1. Lee S, Hahn S. Nature (London) 1995;376:609–612. - PubMed
    1. Price M A, Tullius T D. Methods Enzymol. 1992;212:194–219. - PubMed
    1. Latham J A, Cech T R. Science. 1989;245:276–282. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources