Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

To incise or not and where: SET-domain methyltransferases know.

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.

Nucleosome remodeling at origins of global genome-nucleotide excision repair occurs at the boundaries of higher-order chromatin structure.

Repair of UV-induced DNA damage requires chromatin remodeling. How repair is initiated in chromatin remains largely unknown. We recently demonstrated that global genome-nucleotide excision repair (GG-NER) in chromatin is organized into domains in relation to open reading frames. Here, we define these domains, identifying the genomic locations from which repair is initiated. By examining DNA damage-induced changes in the linear structure of nucleosomes at these sites, we demonstrate how chromatin remodeling is initiated during GG-NER. In undamaged cells, we show that the GG-NER complex occupies chromatin, establishing the nucleosome structure at these genomic locations, which we refer to as GG-NER complex binding sites (GCBSs). We demonstrate that these sites are frequently located at genomic boundaries that delineate chromosomally interacting domains (CIDs). These boundaries define domains of higher-order nucleosome-nucleosome interaction. We demonstrate that initiation of GG-NER in chromatin is accompanied by the disruption of dynamic nucleosomes that flank GCBSs by the GG-NER complex.

Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin.

The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome-NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome.

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.

Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq.

Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical's DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.

DNA replication initiation shapes the mutational landscape and expression of the human genome.

The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity. We demonstrate that these replication initiation-dependent mutational processes exert an influence on phenotypic diversity in humans that is disproportionate to the origins' genomic size: By increasing mutational loads at gene promoters and splice junctions, the presence of an origin significantly influences both gene expression and mRNA isoform usage. Last, we show that mutagenesis at origins not only drives the evolution of origin sequences but also contributes to sculpting regulatory domains of the human genome.

The NER protein Rad33 shows functional homology to human Centrin2 and is involved in modification of Rad4.

In the yeast Saccharomyces cerevisiae the Rad4-Rad23 complex is implicated in the initial damage recognition of the Nucleotide Excision Repair (NER) pathway. NER removes a variety of lesions via two subpathways: Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). We previously showed that the new NER protein Rad33 is involved in both NER subpathways TCR and GGR. In the present study we show UV induced modification of Rad4 that is strongly increased in cells deleted for RAD33. Modification of Rad4 in rad33 cells does not require the incision reaction but is dependent on the TCR factor Rad26. The predicted structure of Rad33 shows resemblance to the Centrin homologue Cdc31. In human cells, Centrin2 binds to XPC and is involved in NER. We demonstrate that Rad4 binds Rad33 directly and via the same conserved amino acids required for the interaction of XPC with Centrin2. Disruption of the Rad4-Rad33 interaction is sufficient to enhance the modification of Rad4 and results in a repair defect similar to that of a rad33 mutant. The current study suggests that the role of Rad33 in the Rad4-Rad23 complex might have parallels with the role of Centrin2 in the XPC-HHR23B complex.

Integrated Microarray-based Tools for Detection of Genomic DNA Damage and Repair Mechanisms.

The genetic information contained within the DNA molecule is highly susceptible to chemical and physical insult, caused by both endogenous and exogenous sources that can generate in the order of thousands of lesions a day in each of our cells (Lindahl, Nature 362(6422):709-715, 1993). DNA damages interfere with DNA metabolic processes such as transcription and replication and can be potent inhibitors of cell division and gene expression. To combat these regular threats to genome stability, a host of DNA repair mechanisms have evolved. When DNA lesions are left unrepaired due to defects in the repair pathway, mutations can arise that may alter the genetic information of the cell. DNA repair is thus fundamental to genome stability and defects in all the major repair pathways can lead to cancer predisposition. Therefore, the ability to accurately measure DNA damage at a genomic scale and determine the level, position, and rates of removal by DNA repair can contribute greatly to our understanding of how DNA repair in chromatin is organized throughout the genome. For this reason, we developed the 3D-DIP-Chip protocol described in this chapter. Conducting such measurements has potential applications in a variety of other fields, such as genotoxicity testing and cancer treatment using DNA damage inducing chemotherapy. Being able to detect and measure genomic DNA damage and repair patterns in individuals following treatment with chemotherapy could enable personalized medicine by predicting response to therapy.

Histone modification and chromatin remodeling during NER.

Here we review our developments of and results with high resolution studies on global genome nucleotide excision repair (GG-NER) in Saccharomyces cerevisiae. Technologies were developed to examine NER at nucleotide resolution in yeast sequences of choice and to determine how these related to local changes in chromatin. We focused on how GG-NER relates to histone acetylation for its functioning and we identified the histone acetyltransferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. Factors influencing this Gcn5-mediated event are considered which include Rad16, a GG-NER specific SWI/SNF factor and the yeast histone variant of H2AZ (Htz1). We describe results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then consider the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the NER of UV-induced cyclobutane pyrimidine dimers throughout entire yeast genome. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage before it is returned to its pre-damaged status to maintain epigenetic codes.