Efficiency of Excision of 8-Oxo-guanine within DNA Clustered Damage by XRS5 Nuclear Extracts and Purified Human OGG1 Protein

Abstract

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.

Induction and Processing of Oxidative Clustered DNA Lesions in56Fe-Ion-Irradiated Human Monocytes

“…Cells in complete medium at an average cell concentration of ϳ10 6 cells ml Ϫ1 were exposed to 56 Fe ions (1.046 GeV/nucleon, LET ϭ 148 keV/m, dose rate 0.6-1.1 Gy min Ϫ1 ) at the AGS facility (Brookhaven National Laboratory, Upton, NY) either (1) for measurement of initial damage levels, chilled on ice for 15 min, irradiated on ice and immediately harvested after irradiation by immersion in liquid nitrogen or (2) for repair measurements, irradiated at room temperature, placed at 37ЊC immediately after irradiation for further incubation, then harvested as described. In repair measurements, medium was changed every 2-3 days.…”

“…We used human monocytes irradiated with 56 Fe ions at doses from 0-10 Gy. Three different E. coli repair proteins were used as enzymatic probes of clustered DNA damage: Fpg (oxidized purines), Endo III (oxidized pyrimidines), and Endo IV (abasic sites).…”

“…DSBs were the most prevalent damage found compared to oxidative clustered DNA lesions. For the first time, information on repair kinetics is presented for oxidative clustered DNA lesions induced by 56 Fe ions in conjunction with data on apoptosis.…”

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

“…In mammalian cells, these lesions are primarily removed by DNA glycosylases and abasic endonucleases [38]. However, when Tg and 8-oxoG occur within the clustered DNA lesions, normal repair by the glycosylases at base excision repair (BER) is often thwarted by adjacent breaks and gaps [39]. As a result, these damaged bases may in some cases require removal by DSB-specific end-trimming mechanisms involving various nucleases discussed in detail below.…”

Repair of Clustered DNA Lesions

“…AP sites or SSBs inhibit the processing of 8-oxoguanine when they are closely located on the opposite DNA strand (11). Thus, in the course of repair, clustered lesions containing an 8-oxoguanine opposed by either another oxidized base or an abasic site (e.g.…”