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Genome-wide mapping of DNA strand breaks.

Leduc F, Faucher D, Bikond Nkoma G, Grégoire MC, Arguin M, Wellinger RJ, Boissonneault G - PLoS ONE (2011)

Bottom Line: Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered.Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution.This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

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Related in: MedlinePlus

Protocol for damaged DNA immunoprecipitation.DNA breaks are end-labeled by the incorporation of biotin-dNTP at 3′OH termini by the terminal transferase. The labeled DNA is further digested in fragments of suitable size for immunoprecipitation. DNA fragments are immunoprecipitated with anti-biotin antibodies and protein G-coated magnetic beads. Enriched DNA fragments can be identified and quantified using qPCR, microarrays or next-generation sequencing (NGS).
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pone-0017353-g001: Protocol for damaged DNA immunoprecipitation.DNA breaks are end-labeled by the incorporation of biotin-dNTP at 3′OH termini by the terminal transferase. The labeled DNA is further digested in fragments of suitable size for immunoprecipitation. DNA fragments are immunoprecipitated with anti-biotin antibodies and protein G-coated magnetic beads. Enriched DNA fragments can be identified and quantified using qPCR, microarrays or next-generation sequencing (NGS).

Mentions: Currently available methods to assess DNA damage include electrophoretic techniques such as pulse-field gel electrophoresis (PFGE)[1] or single-cell electrophoresis (Comet assay)[2] for a global assessment of DNA fragmentation. Ligation-mediated polymerase chain reaction (LM-PCR) is also commonly used for quantitatively displaying DNA lesions in mammalian cells because it combines nucleotide-level resolution with the sensitivity of PCR[3] but is limited by the use of sequence-specific primers. All of the aforementioned approaches suffer from one important limitation as they they do not allow mapping of DNA strand breaks on a genome-wide scale and cannot identify new sensitive sites or hotspots harboring such break sites. Therefore, a reproducible method for the genome-wide mapping of DNA strand breaks would be useful to study their global distribution all at once and monitor any alteration in damage profile under different experimental conditions. Here, we provide a detailed description of a straightforward strategy, termed “damaged DNA immunoprecipitation” or “dDIP”. This method uses the immunoprecipitation of biotin-modified nucleotides added by the terminal deoxynucleotidyl transferase (TdT) at sites of DNA damage (see Figure 1). Although a similar approach has been used recently to map nuclear receptor-dependant tumor translocations [4], we describe for the first time its genome-wide application resulting from the development and optimization of this method by our group over the past three years. Because of its potential widespread use in genome research, we provide the important experimental details and key findings for the reliable capture and enrichment of damaged DNA sequences in the form of strand breaks.


Genome-wide mapping of DNA strand breaks.

Leduc F, Faucher D, Bikond Nkoma G, Grégoire MC, Arguin M, Wellinger RJ, Boissonneault G - PLoS ONE (2011)

Protocol for damaged DNA immunoprecipitation.DNA breaks are end-labeled by the incorporation of biotin-dNTP at 3′OH termini by the terminal transferase. The labeled DNA is further digested in fragments of suitable size for immunoprecipitation. DNA fragments are immunoprecipitated with anti-biotin antibodies and protein G-coated magnetic beads. Enriched DNA fragments can be identified and quantified using qPCR, microarrays or next-generation sequencing (NGS).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045442&req=5

pone-0017353-g001: Protocol for damaged DNA immunoprecipitation.DNA breaks are end-labeled by the incorporation of biotin-dNTP at 3′OH termini by the terminal transferase. The labeled DNA is further digested in fragments of suitable size for immunoprecipitation. DNA fragments are immunoprecipitated with anti-biotin antibodies and protein G-coated magnetic beads. Enriched DNA fragments can be identified and quantified using qPCR, microarrays or next-generation sequencing (NGS).
Mentions: Currently available methods to assess DNA damage include electrophoretic techniques such as pulse-field gel electrophoresis (PFGE)[1] or single-cell electrophoresis (Comet assay)[2] for a global assessment of DNA fragmentation. Ligation-mediated polymerase chain reaction (LM-PCR) is also commonly used for quantitatively displaying DNA lesions in mammalian cells because it combines nucleotide-level resolution with the sensitivity of PCR[3] but is limited by the use of sequence-specific primers. All of the aforementioned approaches suffer from one important limitation as they they do not allow mapping of DNA strand breaks on a genome-wide scale and cannot identify new sensitive sites or hotspots harboring such break sites. Therefore, a reproducible method for the genome-wide mapping of DNA strand breaks would be useful to study their global distribution all at once and monitor any alteration in damage profile under different experimental conditions. Here, we provide a detailed description of a straightforward strategy, termed “damaged DNA immunoprecipitation” or “dDIP”. This method uses the immunoprecipitation of biotin-modified nucleotides added by the terminal deoxynucleotidyl transferase (TdT) at sites of DNA damage (see Figure 1). Although a similar approach has been used recently to map nuclear receptor-dependant tumor translocations [4], we describe for the first time its genome-wide application resulting from the development and optimization of this method by our group over the past three years. Because of its potential widespread use in genome research, we provide the important experimental details and key findings for the reliable capture and enrichment of damaged DNA sequences in the form of strand breaks.

Bottom Line: Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered.Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution.This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Show MeSH
Related in: MedlinePlus