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. 2018:1672:155-166.
doi: 10.1007/978-1-4939-7306-4_13.

Mapping DNA Breaks by Next-Generation Sequencing

Laura Baranello  1   2 , Fedor Kouzine  3 , Damian Wojtowicz  3 , Kairong Cui  4 , Keji Zhao  4 , Teresa M Przytycka  5 , Giovanni Capranico  6 , David Levens  3
Affiliations

Mapping DNA Breaks by Next-Generation Sequencing

Laura Baranello et al. Methods Mol Biol. 2018.

Abstract

Here, we present two approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) in the genome of human cells. We named these methods respectively DSB-Seq and SSB-Seq. We tested the DSB and SSB-Seq in HCT1116, human colon cancer cells, and validated the results using the topoisomerase 2 (Top2)-poisoning agent etoposide (ETO). These methods are powerful tools for the direct detection of the physiological and pathological "breakome" of the DNA in human cells.

Keywords: DNA damage; Double-strand breaks (DSBs); Etoposide (ETO); Single-strand breaks (SSBs); Topoisomerase 2 (Top2).

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Figures

Fig. 1
Fig. 1
Types of DNA damage. Exogenous and endogenous DNA damaging agents generate various types of lesions including SSBs and DSBs. PARP predominantly acts as a sensor of SSB [17]. RPA binds to regions of single-stranded DNA (ssDNA) that are exposed to stalled replication forks or after DSB resection [18]. The multifunctional MRN complex and KU detect DSBs, FANCM is required for the DNA interstrand crosslink (ICL)-induced checkpoint response [19]. FANCM = Fanconi anemia complementation group M; ICL = interstrand crosslink; MRN = MRE11-RAD50-NBS1 complex; PARP = poly(ADP-ribose) polymerase; RPA = replication protein A
Fig. 2
Fig. 2
DNA breaks mapping workflow. (a) SSBs are labeled during nick translation using nucleotides covalently linked to digoxigenin (blue circle). The DNA is subsequently purified, sonicated and incubated with anti-digoxigenin antibody (anti-DIG). The immuno-precipitated DNA is sequenced. (b) 3′ tails of DSBs are ligated to biotinylated nucleotides (red circle). After sonication the labeled fragments are captured on streptavidin beads (pink circle). Tails are removed from released fragments and DNA is sequenced
Fig. 3
Fig. 3
Representative example of High Molecular Weight (HMW) DNA after the purification steps described in Subheading 3.1. In lanes 1 and 2 we run two different markers. The numbers on the left refer to the molecular weight of the marker in lane 2
See this image and copyright information in PMC

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