Spontaneous DNA Damage Stimulates Topoisomerase II-mediated DNA Cleavage

Abstract

Apurinic sites are position-specific poisons of topoisomerase II and stimulate DNA scission ϳ10 -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.The integrity of the genetic material is constantly challenged by events that generate damage in DNA (1-3). An important source of this damage originates from spontaneous alterations within the double helix such as the loss of bases (i.e. generation of apurinic or apyrimidinic sites) and the deamination of cytosine residues (i.e. conversion of cytosine to uracil) (1, 3).Of all spontaneous DNA lesions, apurinic sites are the most commonly formed (1, 3). It is estimated that ϳ10,000 apurinic sites are created per mammalian cell on a daily basis (4). Although rates of spontaneous pyrimidine hydrolysis and cytosine deamination in duplex DNA are ϳ20-and 70-fold lower than that of purine loss (3), respectively, these lesions are frequently induced by exogenous mutagens such as UV irradiation, oxidation, and DNA-reactive chemicals (1, 3, 5-7).In addition to the reported mutagenic properties of apurinic/ apyrimidinic sites and deaminated cytosines (1, 3, 5-7), the induction of spontaneous damage in DNA by heat-acid treatment also enhances the formation of topoisomerase II-cleaved DNA complexes ϳ6-fold in vitro (8). These cleavage complexes are normally short-lived intermediates in the catalytic cycle of the enzyme and are tolerated by the cell (9 -12). However, conditions that shift the cleavage/religation equilibrium of the enzyme toward the cleaved state "poison" topoisomerase II and trigger numerous mutagenic events, such as insertions, deletions, illegitimate recombination, and potentially, cancer-related chromosomal translocations (13-19). When topoisomerase II-mediated DNA breaks are present in sufficient numbers, they initiate a programmed series of events that ultimately culminates in cell death (20 -23).The potentially lethal nature of topoisomerase II has been exploited by seve...

Cytosine Arabinoside Lesions Are Position-specific Topoisomerase II Poisons and Stimulate DNA Cleavage Mediated by the Human Type II Enzymes

“…Substitution of a ␤-hydroxyl for a hydrogen at the 2Ј-position of deoxycytosine (converting the deoxyribose ring to an arabinose) stimulated DNA scission mediated by the ␣ and ␤ isoforms of human topoisomerase II but did so with a positional specificity that differed from those of other DNA lesions. Moreover, in contrast to previous findings with abasic sites (26), additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings suggest sugar ring modifications can alter topoisomerase II function and raise the possibility that the enzyme may mediate some of the physiological effects of araC.…”

ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis

“…AP sites are also formed by hydrolytic decay of DNA, which is estimated to produce around 10,000 AP sites per day in each mammalian cell (3). AP sites are cytotoxic (35,50), and as they lack genetic information, mutagenic (2,22,40).…”

Reactive Oxygen Species Elicit Apoptosis by Concurrently Disrupting Topoisomerase II and DNA-Dependent Protein Kinase

“…These data also suggest that the induced ROS may act as signaling molecules rather than directly breaking the DNA duplex. Several previous studies have identified various mechanisms involved in the activation of Topo II-mediated DNA cleavage, including DNA structural modification, enzyme modification, acidic pH environment, and oxidative stress (Zechiedrich et al, 1989;Kingma and Osheroff, 1997;Li et al, 1999;Wang et al, 2001). The thiol-containing cysteine residue, generally as a catalytic element in the active domains of enzymatic proteins, can be oxidatively modified by ROS, thus leading to enzyme inactivation (Michiels et al, 2002).…”