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

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

(doi: 10.3390/genes9120632)

Additionally, chromatin looping involved in 3D genome organization and gene regulation is increasingly recognized as a possible contributor to DSB events.

This review;

1.mechanisms of endogenous DSB formation

2.genome-wide profiling of DSB

Future direction on this field -- genome-wide DSB formation and repair.

DSBs can trigger cell death or give rise to structural genomic rearrangements associated with carcinogenesis and other diseases. The frequency of DSBs is estimated to be 10–50 events per cell per day.

in healthy individuals the majority of DSBs is thought to originate from within the nucleus, where DSBs form during fundamental processes such as DNA replication, meiosis, antibody diversification, gene transcription, and—although indirectly—cellular metabolism.

DNA transactions—especially during DNA replication, transcription, and 3D genome folding.

Replication forks are forced to slow or stall in a global manner when resources required for faithful completion of replication run out.

Activated oncogenes can perturb replication timing by increasing origin firing -- leading to unligated Okazaki fragments into single-ended DSBs.

oncogene activation can also provoke premature cellular senescence in a process called oncogene-induced senescence, which has been uncovered as an important tumor-suppressing strategy in premalignant cells.

Transcription bubbles present a natural obstacle for the replication fork, and consequences of so-called transcription-replication conflicts.

Both transactions use the same DNA template, and collisions are inevitable, especially for long genes that require more than one interphase to be transcribed.

While TRCs may be rare in healthy human cells, their frequency increases when replication timing is perturbed and in situations in which the transcription machinery lingers.

Two transcriptional intermediates that have been implicated in TRCs are DNA/RNA hybrids known as R-loops and backtracked RNAPII complexes.

sensing of replication stress leads to activation of the replication checkpoint, temporary cell cycle arrest, and orchestration of the DDR.

Stalled replication forks are predominantly stabilized by components of the HR repair pathway, which also protect nascent DNA at the stalled fork and support repair of DSBs induced by replication stress.

Transcription-coupled nucleotide excision repair is thought to assist removal of obstacles that cause replication stress such as R-loops, bulky lesions, and arrested transcription complexes.

restart of the replication fork is believed to occur with the help of HR factors and via repriming mediated by local ssDNA, although the precise underlying fork remodeling and reversal mechanisms remain to be elucidated further.

DSBs associated with replication stress occur at higher frequency in certain regions of the genome such as common fragile sites (CFSs).

CFSs are largely cell type-specific, overlap with recurrent copy number alterations (CNAs) seen in cancer—especially with large deletions—and contain genes with presumed tumor-suppressor activity.

In contrast to CFSs, early replicating fragile sites (ERFSs) represent a different class of fragile genomic regions that replicate early, reside in gene-rich accessible chromatin, and overlap with sites of recurrent translocations and rearrangements observed in cancer.

point mutation rates are generally higher in late-replicating genomic regions across eukaryotic species.

Regions with early versus late replication timing exhibit roughly mirroring patterns of DNA accessibility, gene activity, and nuclear positioning, in line with A/B compartmentalization.

The more permissive state of chromatin in early-replicating regions likely allows efficient replication, whereas late-replicating—and oftentimes more compact—chromatin requires more origins and thus shorter inter-origin distances.

transcription is in itself, in a replication-independent manner, considered to be a source of DSBs.

the transient strand separation during transcription is thought to render the non-transcribed strand particularly vulnerable.

The genome-wide frequency of DSBs is not only elevated in CFSs, ERFSs, and long, late-replicating

genes, but also in accessible chromatin and, in particular, near transcriptionally active genes, with a significant enrichment around the transcription start site (TSS).

in estrogen-responsive breast cancer cells, DSBs form at estrogen-responding genes through base excision repair (BER), aimed at repairing cytosines deaminated by the action of the APOBEC3B enzyme, which is frequently deregulated in cancer cells.

TOP2-induced DSBs have been suggested to be needed for the transcription of some or all genes. TOP2B-induced DSBs in gene promoters to be required for transcription activation upon hormone stimulation. TOP2 forms transient DSBs that help resolve positive DNA supercoiling, which builds up ahead of transcription and replication forks, halting their progression or leading to strand breaks. Although the precise mechanism underlying DSB-induced transcription activation remains elusive, TOP2-mediated release of the topological stress brought about by supercoils may be sufficient to stimulate RNAPII processivity.

TOP2 in removing supercoils, its activity is a double-edged sword. Despite its successful use in chemotherapy, etoposide has also been associated with the emergence of therapy-related secondary acute myeloid leukemias driven by recurrent genomic translocations between regions coinciding with etoposide-induced DSBs. how transcription activation frequently involves the formation of a transient DSB by TOP2.

elevated DSB susceptibility at genomic regions involved in 3D genome organization. TOP2B has been identified at cis-regulatory genomic elements bound by CCCTC-binding factor (CTCF) or by both CTCF and cohesin. These TOP2B/CTCF/cohesin-bound elements overlap regions uncovered to be relevant for 3D genome organization, such as the borders or anchors of transcriptionally active supercoiling domains and TADS, as well as the elements involved in regulatory chromatin loops.

While the levels of etoposide-induced DSBs correlate with gene expression levels, TOP2B-induced DSBs have been reported to be largely transcription-, replication-, and cell type-independent, and instead related to the action of TOP2B at the border of chromatin loops.

frequency of TOP2-induced DSBs enriched at CTCF/cohesin bound loop anchors correlates with expression levels and directionality of coinciding highly transcribed genes—such as those frequently involved in oncogenic translocations in leukemias. inhibition of transcription elongation led to a decrease in DSB levels across transcribed regions and reduced formation of gene fusions, altogether suggesting that both transcription and 3D chromatin folding contribute to TOP2-related genomic instability.

As torsional stress can contribute to transcription regulation, supercoiling,and knots emerging during chromatin looping could be involved in, for instance, safeguarding timely termination of transcription, transcription direction, or seclusion of regulatory effects.

programmed DSBs in specific gene classes or during specific moments in development, for example, meiotic recombination, and lymphocyte-specific processes V(D)J recombination, the dependency on programmed DSBs poses oncogenic risks.

the DDR can in principle repair programmed and accidental DSBs quickly and faithfully, and rescue stalled replication forks to prevent their collapse, DSBs are drivers of most of the structural genomic rearrangements observed in human (cancer) genomes.

During early carcinogenesis, increasing levels of DSBs—due to enhanced replication stress and transcriptional rewiring—can exhaust the DDR and eventually compromise faithful DSB repair.

Copy number variation (CNV) is widespread in the human genome and underlies natural variation and evolution, but also cancer and developmental and neurological disorders.

In C-NHEJ, DSB ends are repaired without the need for homology, and although the resulting junctions are mostly accurate or have small deletions, free DNA may be inserted or translocations can be formed.

In contrast, MMEJ—the best-known a-EJ pathway—joins DSB ends based on microhomology (<25 nt), mediated by the error-prone DNA polymerase theta, which is frequently upregulated in cancer. most of the mechanisms underlying copy number gains involve replication-based mechanisms, segment amplifications can also arise in a non-replicative manner via breakage-fusion-bridge (BFB) cycles.

patterns of single nucleotide variants (SNVs) have helped uncover distinct mutational signatures in cancer genomes, which has led to improved understanding of the underlying causative processes in certain cancer types.

similar approach is applied to multi-nucleotide structural rearrangements or copy number changes in cancer genomes, based on the identification of junctions between genomic sequences that are not naturally together in the reference genome. the computational analyses required to identify complex rearrangement patterns are challenging, successful reconstruction of the genomic junctions in a cancer genome can reveal repair signatures composed of gains, losses, amplifications and rearrangements. As certain repair signatures are largely cancer type-specific, they have been harnessed to classify cancers and improve stratification of specific cancer subtypes, as well as to study clonal relationships among metastases and the corresponding primary tumors. Although genome-wide profiling of structural rearrangements enables investigation of repair signatures and errors—based on identification of non-linear junctions—these approaches typically reveal past events that have occurred at some point in the history of the cell.

Various methods for genome-wide DSB detection and identification have been developed, with the aim of obtaining insight into genome fragility and its molecular basis.

The first genome-wide DSB landscape was mapped in yeast, using chromatin immunoprecipitation (ChIP) on a tiled microarray (ChIP-chip) based on antibodies against the phosphorylated form of histone variant H2AX, gH2AX. revealed H2AX enrichment at loci prone to replication fork stalling and breakage, of which half mapped to repressed protein-coding genes. More recently, ChIP-seq for TOP2B binding was used in addition to H2AX to indirectly detect DSBs in NSPCs upon exogenous activation. This revealed that TOP2B-dependent DSBs accumulate in the promoter region of early-response genes and are required for their transcription. gH2A.X not only accumulates around DSB sites, but is also recruited to regions with SSBs and to sites undergoing nucleotide excision repair in G1.

The first method to directly capture and identify DSBs in fixed cells in situ was Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS). BLESS was applied to identify endogenous and replication stress-induced DSBs, and to determine the specificity of CRISPR endonucleases. Although BLESS does not depend on transfection or NHEJ repair, its labor-intensive protocol requires large amounts of input material and cell fixation, which has been related to the observed high background levels of DSBs.

Since the structure of DSB ends is believed to affect DSB repair pathway choice, DSB identification approaches that apply DSB end blunting prior to adapter ligation, and especially those that apply formaldehyde fixation, are at risk of altering the original and potentially informative structure of DSB ends.

To specifically study DSB end structures and to unravel the activities of DSB repair pathways in protecting DSB ends, hairpin capture of DNA end structures (HCoDES) was developed, in which DSB ends are treated with ssDNA ligase to form hairpins that allow PCR-based amplification and subsequent sequencing to analyze the precise 50 end and 30 end position of both strands.

Ultimately, assay choice should be steered by the specific research question and the characteristics of the sample to be profiled.

Fragility scores can for example be correlated to maps of replication timing, transcriptional activity, or chromatin accessibility, preferentially generated in parallel on the same cell type. the frequency of DSB events can be analyzed in light of genome-wide maps of R-loops generated with DNA-RNA immunoprecipitation combined with sequencing (DRIP-seq), binding of transcription or architectural factors created with (exo-)ChIP-seq, and 3D genome folding or nuclear organization, for instance assessed with (capture) Hi-C, or DamID technology.

While the genome-wide DSB frequency, distribution, and repair rate differ between underlying causative processes, these will in turn also be affected by the spatiotemporally varying architecture of the genome and cell cycle dynamics. many factors associated with genome-wide fragility, including DNA replication and transcription, are inherently interconnected, and correlate to underlying genomic and epigenomic features across multiple genomic scales, such as gene density, GC levels, and DNase I hypersensitivity.

In addition to certain genomic regions being more prone than others to form DSBs under specific conditions, the possibility of compartmentalized DSB repair and the sequestering of some DSBs to dedicated nuclear regions in mammalian cells is now emerging. DSB repair compartmentalization might allow for spatiotemporal uncoupling of different DSB repair pathways in a cell cycle phase-dependent manner, potentially acting to suppress genomic rearrangements between juxtaposed repetitive chromosomal domains. the choice of DSB repair pathway is controlled by multiple factors, including cell cycle phase and proliferative state, as well as local chromatin composition, and by the severity and nature of the DSBs. future studies of repair pathway choice, rate, and fidelity.

the effects on fragility are generally not homogenous throughout the cell population, which makes it challenging to draw general conclusions about their effects on transcription or repair. No method that allows for 100% efficiency of DSB induction at defined sites in the human genome. that the cellular response to CRISPR/Cas9-induced DSBs may not be indicative of regular repair of endogenous DSBs. In line with this, also the events observed at the AsiSI sites introduced in DIvA cells may not fully recapitulate the same processes that occur upon endogenous DSB formation. Yet, both of these approaches are highly valuable to broaden our knowledge of DSB biology in the context of the nucleus.

DSB profiling is applied to endogenous or exogenous DSBs, the effect of noise—as well as the absence of a proper notion of what may be considered as background signal—is hard to work around, and low-abundance yet recurrent or pathological DSBs may be masked. exploring the levels of ongoing genome fragility by mapping DSBs directly in clinical specimens might provide new insights into the mechanisms that shape cancer genomes and their evolution. DSB landscapes may hold clues on highly fragile regions that underlie rearrangements frequently associated with a tendency to metastasize. comparing DSB profiles with the landscape of rearrangements at a later timepoint during tumor evolution, as recently attempted, will be instrumental for a better understanding of how DSBs are converted into rearrangements that fuel tumor progression.

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