Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand

Abstract

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation

“…However, middle age (8 months) and old age (24 months) animals do accumulate significant amounts of oxidative DNA lesions, mostly in the transcribed but not in the nontranscribed genes of various tissues, further implicating NEIL2's biological role in TC-BER. Two other reports also have indicated transcription-coupled repair of oxidized bases in mammalian cells, as well as in yeast (59,60). To our knowledge, ours is the first in vivo evidence for such repair of oxidized bases in mammals.…”

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

“…The data shown in Figure 4 indicate that repair of 8-oxoG takes place with relatively slow kinetics, whereas several reports state rapid repair with nearly 50% of the lesions removed rapidly after being induced (11,32). The design of our experiments included a 30-min incubation of the cells after removal of potassium bromate, to allow repair of frank single-strand breaks, and thus reduce the background of DNA in comet tails.…”

“…Recently developed methods use PCR amplification to detect oxidative base damage in DNA with high sensitivity. However, they cannot distinguish the specific type of DNA damage induced by ROS (11), and they do not permit examination of repair at the strand-specific level (12). …”

“…Thus, to study repair of low, physiologically relevant levels of specific oxidized DNA damage, new methodologies with high sensitivity and specificity are needed. Although it has been suggested that oxidation damage in DNA is repaired in a transcription-dependent manner (11,12,16), direct and reproducible evidence of preferential repair of specific oxidized DNA bases in the transcribed strands of active genes has been lacking.…”

Transcription-coupled repair: an update

“…Several groups have presented direct and indirect evidence of preferential repair of oxidized bases (Banerjee et al 2010; Reis et al 2012; Spivak and Hanawalt 2006). Menoni et al developed a novel laser-directed method to inflict oxidative DNA damage locally in living cells and observed a clear transcription-dependent recruitment of CSB to these lesions (Menoni et al 2007); they have subsequently documented the localization of XPC, but not of XPA or XPB, to oxidatively-damaged spots, which suggests that “canonical NER” is not involved in genomic repair of those lesions (Menoni et al 2012).…”