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

António M C Reis, Wilbur K. Mills, Ilangovan Ramachandran, Errol C. Friedberg, David Thompson, Lurdes Queimado

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)206-219
Number of pages14
JournalNucleic Acids Research
Volume40
Issue number1
DOIs
StatePublished - Jan 2012

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DNA Repair
DNA Damage
Nucleotides
Yeasts
Dermatoglyphics
Saccharomyces cerevisiae
Real-Time Polymerase Chain Reaction
Mutation
Growth

ASJC Scopus subject areas

  • Genetics

Cite this

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand. / Reis, António M C; Mills, Wilbur K.; Ramachandran, Ilangovan; Friedberg, Errol C.; Thompson, David; Queimado, Lurdes.

In: Nucleic Acids Research, Vol. 40, No. 1, 01.2012, p. 206-219.

Research output: Contribution to journalArticle

Reis, António M C ; Mills, Wilbur K. ; Ramachandran, Ilangovan ; Friedberg, Errol C. ; Thompson, David ; Queimado, Lurdes. / Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 1. pp. 206-219.
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