Quantification of 8-oxoG in Plant Telomeres

Abstract

Chemical modifications in DNA impact gene regulation and chromatin structure. DNA oxidation, for example, alters gene expression, DNA synthesis and cell cycle progression. Modification of telomeric DNA by oxidation is emerging as a marker of genotoxic damage and is associated with reduced genome integrity and changes in telomere length and telomerase activity. 8-oxoguanine (8-oxoG) is the most studied and common outcome of oxidative damage in DNA. The G-rich nature of telomeric DNA is proposed to make it a hotspot for oxidation, but because telomeres make up only a tiny fraction of the genome, it has been difficult to directly test this hypothesis by studying dynamic DNA modifications specific to this region in vivo. Here, we present a new, robust method to differentially enrich telomeric DNA in solution, coupled with downstream methods for determination of chemical modification. Specifically, we measure 8-oxoG in Arabidopsis thaliana telomeres under normal and oxidative stress conditions. We show that telomere length is unchanged in response to oxidative stress in three different wild-type accessions. Furthermore, we report that while telomeric DNA comprises only 0.02–0.07% of the total genome, telomeres contribute between 0.2 and 15% of the total 8-oxoG. That is, plant telomeres accumulate 8-oxoG at levels approximately 100-fold higher than the rest of the genome under standard growth conditions. Moreover, they are the primary targets of further damage upon oxidative stress. Interestingly, the accumulation of 8-oxoG in the chromosome body seems to be inversely proportional to telomere length. These findings support the hypothesis that telomeres are hotspots of 8-oxoG and may function as sentinels of oxidative stress in plants.