Endogenous DNA Double-Strand Breaks during DNA Transactions: Emerging Insights and Methods for Genome-Wide Profiling

Abstract

DNA double-strand breaks (DSBs) jeopardize genome integrity and can-when repaired unfaithfully-give rise to structural rearrangements associated with cancer. Exogenous agents such as ionizing radiation or chemotherapy can invoke DSBs, but a vast amount of breakage arises during vital endogenous DNA transactions, such as replication and transcription. Additionally, chromatin looping involved in 3D genome organization and gene regulation is increasingly recognized as a possible contributor to DSB events. In this review, we first discuss insights into the mechanisms of endogenous DSB formation, showcasing the trade-off between essential DNA transactions and the intrinsic challenges that these processes impose on genomic integrity. In the second part, we highlight emerging methods for genome-wide profiling of DSBs, and discuss future directions of research that will help advance our understanding of genome-wide DSB formation and repair.

Keywords: 3D genome structure; DNA double-strand breaks; cancer; chromatin looping; genome-wide methods; rearrangements; replication; topoisomerases; transcription.

Figure 1

Endogenous DNA double-strand breaks (DSBs) during DNA transactions. (A) Left panel: during DNA replication, pre-existing DNA lesions such as G-quadruplexes and inter-strand crosslinks invoke replication stress and cause replication fork stalling. Right panel: an example of how intermediate structures during fork remodeling can lead to formation of a single-ended DSB. Bottom: replication through a single-strand break or nick can result in a single-ended DSB [36,39,41]. (B) Transcription complexes obstruct replication fork progression, particularly when transcription-replication encounters are head-on (top) [68], when the nascent RNA has formed an R-loop that stabilizes the RNAPII association with the DNA (middle) [62], and when the RNAPII complex is paused and displays backtracking (bottom, arrow indicates backward sliding of the RNAPII) [59]. (C) Transcription-related activity-induced DSBs emerge at sites of topoisomerase 2 (TOP2) action [56,71], which is required to release positive (+) supercoiling building up ahead of the RNAPII complex. TOP2-mediated DSBs enable transcription but can also lead to non-resolved DSBs when repair is escaped or fails [72]. (D) Genomic regions involved in 3D genome looping experience torsional stress that requires TOP2 activity to be resolved [73]. As a result, chromatin loop boundaries or anchors may accumulate TOP2-dependent DSBs [74].

Figure 2

Methods for genome-wide DSB profiling. (TOP) Proteins recruited to DSB sites—or associated with DSB formation—serve as a proxy for DSB formation. Chromatin containing the protein of choice is pulled down, and the extracted DNA—representing the underlying genomic regions—can then be analyzed by microarray (ChIP-chip) or high-throughput sequencing (ChIP-seq) [183,187]. The resolution of the generated binding profiles typically depends on the chosen protein. (RIGHT) Methods for in vivo capturing of DSBs utilize the non-homologous end-joining (NHEJ) repair machinery of the cell to either incorporate short dsDNA oligos (ODN) (in genome-wide unbiased identification of DSBs enabled by sequencing, GUIDE-seq [194]), or integration-deficient lentiviral vectors (in IDLV capture [195,196]) at the genomic sites of DSBs, or to generate translocation junctions between emerging DSB ends and a bait DSB, exogenously introduced and then induced in the cell (translocation-capture sequencing (TC-Seq) and high-throughput genome-wide translocation mapping (HTGTS) or linear amplification-mediated (LAM)-HTGTS, and derived methods [197,198,199,200]). Afterwards, cells are lysed and DNA is isolated, followed by method-specific approaches for specific amplification or capture of integration or translocation junctions. Subsequently, sequencing libraries are prepared, and sequence reads are aligned to the genome, typically revealing breakpoint clusters genome-wide. (LEFT) In vitro methods for genome-wide DSB identification directly label DSB ends with a dedicated adapter—with or without prior DSB end processing—in fixed cells immobilized on a surface (Breaks Labeling In Situ and Sequencing, BLISS [201]) or fixed cell suspensions (Breaks Labeling, Enrichment on Streptavidin, and Sequencing, BLESS [122]), in unfixed cells embedded in agarose plugs or beads (END-seq [202] and i-BLESS [203], respectively), in isolated DNA (DSB-Seq [126]), or isolated DNA in agarose plugs (Break-seq [204]). After labeling, DSB ends are selectively linearly amplified by in vitro transcription enabled by the BLISS adapter in BLISS. In the other methods, DSB ends are captured onto streptavidin beads that selectively capture the biotin-labeled DSB ends, and then amplified. Finally, sequencing libraries are prepared and the resulting mapped sequence reads reveal single DSB ends distributed genome-wide.