Effects Of Abasic Sites Research Articles

Effects of abasic sites on structural, thermodynamic and kinetic properties of quadruplex structures

Abasic sites represent the most frequent lesion in DNA. Since several events generating abasic sites concern guanines, this damage is particularly important in quadruplex forming G-rich sequences, many of which are believed to be involved in several biological roles. However, the effects of abasic sites in sequences forming quadruplexes have been poorly studied. Here, we investigated the effects of abasic site mimics on structural, thermodynamic and kinetic properties of parallel quadruplexes. Investigation concerned five oligodeoxynucleotides based on the sequence d(TGGGGGT), in which all guanines have been replaced, one at a time, by an abasic site mimic (dS). All sequences preserve their ability to form quadruplexes; however, both spectroscopic and kinetic experiments point to sequence-dependent different effects on the structural flexibility and stability. Sequences d(TSGGGGT) and d(TGGGGST) form quite stable quadruplexes; however, for the other sequences, the introduction of the dS in proximity of the 3′-end decreases the stability more considerably than the 5′-end. Noteworthy, sequence d(TGSGGGT) forms a quadruplex where dS does not hamper the stacking between the G-tetrads adjacent to it. These results strongly argue for the central role of apurinic/apyrimidinic site damages and they encourage the production of further studies to better delineate the consequences of their presence in the biological relevant regions of the genome.

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions.

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIalpha and beta were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N(6)-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage approximately 10-fold. 1,N(6)-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.

Abasic Sites Stimulate Double-stranded DNA Cleavage Mediated by Topoisomerase II: DNA LESIONS AS ENDOGENOUS TOPOISOMERASE II POISONS

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.

Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases.

Abasic sites are thought to be the most frequently occurring cellular DNA damage and are generated spontaneously or as the result of chemical or radiation damage to DNA. In contrast to the wealth of information that exists on the effects of abasic sites on DNA polymerases, very little is known about how these lesions interact with RNA polymerases. An in vitro transcription system was used to determine the effects of abasic sites and single-strand breaks on transcriptional elongation. DNA templates were constructed containing single abasic sites or nicks placed at unique locations downstream from two different promoters and were transcribed by SP6 and Escherichia coli RNA polymerases. SP6 RNA polymerase is initially stalled at abasic sites with subsequent, efficient bypass of these lesions. E. coli RNA polymerase also bypassed abasic sites. In contrast, single-strand breaks introduced at abasic sites completely blocked the progression of both RNA polymerases. Sequence analysis of full-length transcripts revealed that SP6 and E. coli RNA polymerases insert primarily, if not exclusively, adenine residues opposite to abasic sites. This finding suggests that abasic sites may be highly mutagenic in vivo at the level of transcription.