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

Jia Guo, Philip C. Hanawalt, Graciela Spivak

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

Original languageEnglish (US)
Pages (from-to)7700-7712
Number of pages13
JournalNucleic Acids Research
Volume41
Issue number16
DOIs
StatePublished - Sep 2013
Externally publishedYes

Fingerprint

Fluorescence In Situ Hybridization
Ataxia Telangiectasia
DNA
Pyrimidine Dimers
DNA Breaks
Comet Assay
RNA Polymerase II
Endonucleases
DNA Repair
Neurodegenerative Diseases
Genes
Fibroblasts
Genome
Sensitivity and Specificity
Polymerase Chain Reaction
Incidence
8-hydroxyguanine
Neoplasms
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells. / Guo, Jia; Hanawalt, Philip C.; Spivak, Graciela.

In: Nucleic Acids Research, Vol. 41, No. 16, 09.2013, p. 7700-7712.

Research output: Contribution to journalArticle

Guo, Jia ; Hanawalt, Philip C. ; Spivak, Graciela. / Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells. In: Nucleic Acids Research. 2013 ; Vol. 41, No. 16. pp. 7700-7712.
@article{06d7a2a1f51e490faf0a4eab0bff75a6,
title = "Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells",
abstract = "Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.",
author = "Jia Guo and Hanawalt, {Philip C.} and Graciela Spivak",
year = "2013",
month = "9",
doi = "10.1093/nar/gkt524",
language = "English (US)",
volume = "41",
pages = "7700--7712",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "16",

}

TY - JOUR

T1 - Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

AU - Guo, Jia

AU - Hanawalt, Philip C.

AU - Spivak, Graciela

PY - 2013/9

Y1 - 2013/9

N2 - Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

AB - Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

UR - http://www.scopus.com/inward/record.url?scp=84886813438&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84886813438&partnerID=8YFLogxK

U2 - 10.1093/nar/gkt524

DO - 10.1093/nar/gkt524

M3 - Article

VL - 41

SP - 7700

EP - 7712

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 16

ER -