Spontaneous DNA Damage Stimulates Topoisomerase II-mediated DNA Cleavage

Paul S Kingma,Neil Osheroff

doi:10.1074/jbc.272.11.7488

Abstract

Apurinic sites are position-specific poisons of topoisomerase II and stimulate DNA scission approximately 10-18-fold when they are located within the 4-base overhang generated by enzyme-mediated cleavage (Kingma, P. S., and Osheroff, N. (1997) J. Biol. Chem. 272, 1148-1155). To determine whether other major forms of spontaneous DNA damage also act as topoisomerase II poisons, the effects of position-specific apyrimidinic sites and deaminated cytosines (i.e. uracil:guanine mismatches) on the type II enzyme were determined. Both of these lesions stimulated topoisomerase II-mediated DNA scission with the same positional specificity as apurinic sites but were less efficacious. Moreover, apurinic sites dominated the effects of apyrimidinic sites in substrates that contained multiple lesions. The differential ability of spontaneous lesions to enhance DNA cleavage did not correlate with either a decreased stability of the double helix or the size of the gap formed by base loss. Rather, it appears to be due (at least in part) to increased rates of religation for substrates containing apyrimidinic sites or deaminated cytosines. These results suggest that several forms of spontaneous DNA damage are capable of acting as endogenous poisons of topoisomerase II.

Highlights

The integrity of the genetic material is constantly challenged by events that generate damage in DNA [1,2,3]
When located within the 4-base stagger generated by enzyme-mediated DNA cleavage in the sequence shown in Fig. 2, these lesions enhanced the DNA scission event of Drosophila topoisomerase II ϳ10 –18fold
Recent evidence indicates that some forms of spontaneous DNA damage, in addition to their effects on replication fidelity [2, 3, 5], enhance double-stranded DNA cleavage mediated by topoisomerase II [8, 27]

Summary

EXPERIMENTAL PROCEDURES

Topoisomerase II was purified from D. melanogaster embryonic Kc cells as described by Shelton et al [28]. For substrates that contained two apyrimidinic sites, selected oligonucleotides were labeled on their 3Ј-termini using Klenow DNA polymerase, [␣-32P]dGTP, and a template oligonucleotide that contained a 5-base 5Ј-overhang (5Ј-GATCC-3Ј) in addition to the 40-base sequence of the bottom strand. These 3Ј-labeled oligonucleotides were subjected to hydrolysis as described above. 150 pmol of double-stranded oligonucleotide was incubated with 2.5 units of uracil DNA glycosylase in 300 ␮l of 10 mM Hepes-HCl, pH 7.9, 0.1 mM EDTA, and 2.5% glycerol for 30 min at 37 °C [32, 33]. The melting temperature was defined as the temperature at which the first order derivative of the melting curve reached its maximum

RESULTS

Spontaneous DNA Damage Stimulates Topoisomerase II Cleavage

Relative cleavage ϩ EDTA ϩ Salt

Bottom strand

DISCUSSION