Efficiency of Incision of an AP Site within Clustered DNA Damage by the Major Human AP Endonuclease

Abstract

A major DNA lesion induced by ionizing radiation and formed on removal of oxidized base lesions by various glycosylases is an apurinic/apyrimidinic site (AP site). The presence of an AP site within clustered DNA damage, induced following exposure to ionizing radiation or radiomimetic anticancer agents, may present a challenge to the repair machinery of the cell, if the major human AP endonuclease, HAP1, does not efficiently incise the AP site. In this study, specific oligonucleotide constructs containing an AP site located at several positions opposite to another damage [5,6-dihydrothymine (DHT), 8-oxoG, AP site, or various types of single strand breaks] on the complementary strand were used to determine the relative efficiency of the purified HAP1 protein in incising an AP site(s) from clustered DNA damage. A base damage (DHT and 8-oxoG) on the opposite strand has little or no influence on the rate of incision of an AP site by HAP1. In contrast, the presence of either a second AP site or various types of single strand breaks, when located one or three bases 3' to the base opposite to the AP site, has a strong inhibitory effect on the efficiency of incision of an AP site. The efficiency of binding of HAP1 to an AP site is reduced by approximately 1 order of magnitude if a single strand break (SSB) is located one or three bases 3' to the site opposite to the AP site on the complementary strand. If the AP site and either a SSB or a second AP site are located at any of the other positions relative to each other, a double strand break may result.

The biological effects induced by high-charged and energy particles and its application in cancer therapy

“…Many evidence clearly demonstrated that complex DNA lesions are more dif2icult for the cellular machinery to repair than are individual damage sites (28)(29)(30)(31)(32)(33) . With synthetic oligonucleotides containing several types of DNA damage, David-Cordonnier et al demonstrated that the ef2iciency of incision of an AP site within a region of clustered DNA damage is signi2icantly reduced by the presence of a second AP site or SSB (28) .…”

“…With synthetic oligonucleotides containing several types of DNA damage, David-Cordonnier et al demonstrated that the ef2iciency of incision of an AP site within a region of clustered DNA damage is signi2icantly reduced by the presence of a second AP site or SSB (28) . Several studies also have shown APs or 8 -oxoguanine (8-OxoG) sites within clustered DNA damage sites are poorly handled by mammalian cell extracts or puri2ied repair DNA damage site which is defined as mul ple lesions within a region of a few nm.…”

“…(B) Low LET radia on (sparsely ionizing radia on) induces randomly isolated damage (26,27) . enzymes (28)(29)(30) . Some types of clustered damage sites may lead to a lethal DSB during attempted repair of the site in E. coli and mammalian cells (31,(34)(35)(36)(37) .…”

Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

“…Alkaline-labile sites (ALS) [60] including AP sites [13,14] lead to DNA strand cleavage at high pH, and alkaline-based SSB assays are at considerable risk of overestimation of SSB. The comet assay performed at pH 12.1-12.5 has been reported to detect SSB without the contamination of artifactual SSB from cleaved ALS; however, this modification has also been reported to reduce the sensitivity of that assay [61].…”

“…SSB is one of the most frequent DNA lesions, and they are produced daily in nuclei during DNA repair processes such as base excision repair (BER) [12]. It is a primary response pathway for the repair of deleterious DNA lesions including non-bulky DNA adducts, apurinic/apyrimidinic (AP) sites [13,14], and SSB. From a chemical point of view, the abasic site is an alkaline-labile residue that leads to strand breaks through beta-and delta-elimination [15].…”