Source Of Genomic Instability Research Articles

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

HGG-22. LINKS BETWEEN M6A RNA METHYLATION, R-LOOPS AND DNA DAMAGE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Paediatric high-grade gliomas (pHGG) are aggressive malignant tumours of the central nervous system that unfortunately carry poor prognoses. R-loops, which are DNA:RNA hybrids, are a known source of genomic instability in eukaryotic cells. The RNA strand of R-loops can be m6A methylated at double-strand break sites in order to facilitate DNA repair. Therefore, we investigated whether dysregulation of m6A methylation and R-loop homeostasis at DNA damage sites deregulates DNA repair pathways in pHGG. METHODS Immunocytochemistry was performed to assess global m6A, R-loop, γH2AX and several DNA repair protein levels in pHGG cell lines compared to human astrocytes. Colocalisation between m6A and R-loops, R-loops and γH2AX, m6A and γH2AX, R-loops and 53BP1, R-loops and PARP1, R-loops and total ATM, and R-loops and phosphorylated ATM was assessed in pHGG cell lines and human astrocytes. RESULTS Immunocytochemistry analysis revealed that levels of global m6A, R-loop, γH2AX and several DNA repair proteins were generally higher in pHGG cell lines, thereby indicating that cellular homeostasis is dysregulated. Furthermore, a high degree of colocalisation between m6A and R-loops was observed. R-loops were found to colocalise with γH2AX, and m6A was found to colocalise with γH2AX, thereby corroborating the notion that m6A methylated R-loops are likely present at DNA damage sites. Finally, R-loops were also found to colocalise with a variety of DNA repair proteins, including 53BP1, PARP1, total ATM, and phosphorylated ATM, where different Pearson’s correlation coefficient values were obtained, thereby suggesting that R-loops associate with DNA repair proteins to different extents. CONCLUSIONS The link between m6A methylation, R-loops and DNA damage in relation to the pathogenesis of pHGG is currently being investigated. Experiments are also being performed where we are investigating m6A readers to see whether their aberrant expression contributes to disease pathogenesis via deregulating DNA repair pathways.

THE LINK BETWEEN M6A RNA METHYLATION AND R LOOPS IN PAEDIATRIC HIGH-GRADE GLIOMAS AND EPENDYMOMAS

Abstract AIMS Paediatric high-grade glioma (HGG) and ependymoma (EPN) are two devastating malignant tumours of the central nervous system that unfortunately carry a poor prognosis. As R loops are a known source of genomic instability in eukaryotic cells and have previously been revealed to undergo m6A RNA methylation, we sought to investigate the potential link between m6A methylation and R loops in relation to the pathogenesis of paediatric gliomas. METHOD Paediatric HGG cell lines including KNS42, GCE62 and SF188, and paediatric EPN cell lines including BXD- 1425EPN and EPN9, were utilised for experimental analysis, in addition to normal human astrocytes. Immuno- cytochemistry was performed to assess m6A and R loop levels in tumour cell lines compared to normal human astrocytes. Moreover, DNA damage levels and the effects of anti-cancer drugs on R loop levels were also examined. RESULTS Immunocytochemistry analysis revealed that overall m6A and R loop levels were higher in tumour cell lines compared to normal human astrocytes. Furthermore, differences in R loop levels were observed as a result of pharmacological and biological manipulation, including reduction in R loop abundance with PARP inhibitors. CONCLUSIONS The link between m6A methylation and R loops in relation to the pathogenesis of paediatric HGG and EPN is being investigated. The results of these experiments will be presented.

Transcription-Replication Conflicts as a Source of Genome Instability.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

DNA Glycosylases Define the Outcome of Endogenous Base Modifications.

Chemically modified nucleic acid bases are sources of genomic instability and mutations but may also regulate gene expression as epigenetic or epitranscriptomic modifications. Depending on the cellular context, they can have vastly diverse impacts on cells, from mutagenesis or cytotoxicity to changing cell fate by regulating chromatin organisation and gene expression. Identical chemical modifications exerting different functions pose a challenge for the cell's DNA repair machinery, as it needs to accurately distinguish between epigenetic marks and DNA damage to ensure proper repair and maintenance of (epi)genomic integrity. The specificity and selectivity of the recognition of these modified bases relies on DNA glycosylases, which acts as DNA damage, or more correctly, as modified bases sensors for the base excision repair (BER) pathway. Here, we will illustrate this duality by summarizing the role of uracil-DNA glycosylases, with particular attention to SMUG1, in the regulation of the epigenetic landscape as active regulators of gene expression and chromatin remodelling. We will also describe how epigenetic marks, with a special focus on 5-hydroxymethyluracil, can affect the damage susceptibility of nucleic acids and conversely how DNA damage can induce changes in the epigenetic landscape by altering the pattern of DNA methylation and chromatin structure.

DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops

Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of γH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.

PARP1 associates with R-loops to promote their resolution and genome stability.

PARP1 is a DNA-dependent ADP-Ribose transferase with ADP-ribosylation activity that is triggered by DNA breaks and non-B DNA structures to mediate their resolution. PARP1 was also recently identified as a component of the R-loop-associated protein-protein interaction network, suggesting a potential role for PARP1 in resolving this structure. R-loops are three-stranded nucleic acid structures that consist of a RNA-DNA hybrid and a displaced non-template DNA strand. R-loops are involved in crucial physiological processes but can also be a source of genome instability if persistently unresolved. In this study, we demonstrate that PARP1 binds R-loops in vitro and associates with R-loop formation sites in cells which activates its ADP-ribosylation activity. Conversely, PARP1 inhibition or genetic depletion causes an accumulation of unresolved R-loops which promotes genomic instability. Our study reveals that PARP1 is a novel sensor for R-loops and highlights that PARP1 is a suppressor of R-loop-associated genomic instability.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer.

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. Bacteria deploy SIM when faced with DNA double-strand breaks in the presence of conditions that elicit an SOS response. SIM employs DinB, the evolutionary precursor to human trans-lesion synthesis (TLS) error-prone polymerases, and results in mutations concentrated around DNA double-strand breaks with an abundance that decays with distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors, non-inherited mutations from 129 normal samples, and acquired mutations in 3 cell line models of stress-induced adaptive mutation. We introduce statistical methods to identify mutational clusters, quantify their shapes and tease out the potential mechanism that produced them. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. Clusters in normal samples occur more often in the same genomic location across samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis. Additionally, the signatures of TLS contribute the most to mutational cluster formation in both patient samples as well as experimental models of SIM. Furthermore, a measure of cluster shape heterogeneity was associated with cancer patient survival with a hazard ratio of 5.744 (Cox Proportional Hazard Regression, 95% CI: 1.824–18.09). Our results support the conclusion that the ancient and evolutionary-conserved adaptive mutation response found in bacteria is a source of genomic instability in cancer. Biological adaptation through SIM might explain the ability of tumors to evolve in the face of strong selective pressures such as treatment and suggests that the conventional ‘hit it hard’ approaches to therapy could prove themselves counterproductive.

Abstract 1520: Replication stress and the inability to repair damaged DNA, the potential “Achilles' heel” of ecDNA+ tumor cells

Abstract Replication stress (RS) is a primary source of genomic instability, tumorigenesis, and cancer progression. RS is defined as an uncoupling of the replicative helicase and DNA polymerase, resulting in long stretches of fragile single stranded DNA (ssDNA) that is prone to damage. Excessive RS can result in replication catastrophe and cell death, which could be leveraged as a therapeutic strategy to treat high-RS cancers. While overexpression of certain oncogenes has been implicated as a driver of RS in cancer, the precise cellular conditions that result in RS have been difficult to predict. Here we demonstrate that high copy number amplification of genes on non-native extrachromosomal DNA (ecDNA) is associated with elevated RS in comparison to the same amplification on linear chromosomes. The ecDNA bearing cells display heightened basal levels of phosphorylated RPA, a hallmark of ssDNA, and decreased replication fork velocities. Consistent with this finding, the ecDNA amplified cells display greater sensitivity to inhibitors of deoxyribonucleotide production relative to cells harboring chromosomal gene amplification and to normal cells. Moreover, ecDNA bearing cells demonstrate a dramatic vulnerability to reduction in the conditionally essential amino acid glutamine, the primary nitrogen source for de novo nucleotide biosynthesis. The enhanced sensitivity to glutamine starvation is directly correlated with a reduction in ecDNA in tumor cells, and a corresponding selection for chromosomally integrated gene amplification in the surviving population. These phenotypes are rescued with nucleoside supplementation but not anaplerotic TCA cycle intermediates, further reinforcing a link between ecDNA and RS biology. Collectively, observations made here support a novel mechanism for oncogene-induced RS shaped by ecDNA driven oncogene amplification, revealing a novel molecular strategy to identify high-RS tumors and a therapeutic approach to target this “Achilles heel” of tumor biology. Citation Format: Sudhir Chowdhry, Salvador Garcia, Nam-Phuong Nguyen, Anthony Celeste, Edison Tse, Snezana Milutinovic, Deepti Wilkinson, Christian Hassig, Shailaja Kasibhatla. Replication stress and the inability to repair damaged DNA, the potential “Achilles' heel” of ecDNA+ tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1520.

A Comparative Assessment of Replication Stress Markers in the Context of Telomerase.

Simple SummaryGenetic alterations such as oncogenic- or aneuploidy-inducing mutations can induce replication stress as a tumor protection mechansim. Previous data indicated that telomerase may ameliorate the cellular responses that induce replication stress. However, the mechanisms how this may occur are still unclear. In order to address this question, the accurate evaluation of replication stress in the presence and absence of telomerase is crucial. Therefore, we used telomerase negative normal human fibroblasts, as well as their telomerase positive counterparts to compare the suitability of three protein markers (pRPA2, γ-H2AX and 53BP1), which were previously reported to accumulate in response to harmful conditions leading to replication stress in cells. In summary, we find that pRPA2 is the most consistent and reliable marker for the detection of replication stress. Further, we demonstrated that the inhibition of the DNA-damage activated ATM and ATR kinases by specific small compounds impaired the accumulation of pRPA2 foci in the absence of telomerase. These data suggest that telomerase rescues the cells from replication stress upon supression of DNA damage induction by modulating the ATM and ATR signaling pathways, and may therefore support tumor formation of genetically unstable cells.Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers—p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)—are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RASG12V (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.

Defective repair of topoisomerase I induced chromosomal damage in Huntington\u2019s disease

Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington’s disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling.

R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1.

Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA–RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.

Detection and Quantification of RNA Modifications on RNA-DNA Hybrids Using SID-UPLC-MS/MS.

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and an unpaired strand of nontemplate DNA that represent a major source of genomic instability and are involved in regulation of several important biological processes in eukaryotic cells. A growing body of experimental evidence suggests that RNA moieties of RNA-DNA hybrids may convey RNA modifications influencing various aspects of R-loop biology. Here we present a protocol for quantitative analysis of RNA modifications on RNA-DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS). Supplemented by other techniques, this method can be instrumental in deciphering the roles of RNA modifications in R-loop metabolism.

Abstract 1269: Pharmacological targeting of transcription-replication conflict leads to anti-cancer efficacy with minimal side effects in preclinical models

Abstract Unchecked growth of cancer cells places simultaneous demands on DNA replication and transcription. Targeting the transcription-replication conflict (TRC), a major source of genome instability, could have important therapeutic implications. However, testing this hypothesis is difficult without agents directly targeting TRC. Here, we report a small molecule ligand (AOH1996) of the replisome component PCNA, which stabilizes interaction between PCNA and the large subunit (rpb1) of RNA polymerase II through the APIM motif, resulting in proteasome-dependent degradation of rpb1 and lethal DNA damages. Orally administrable, AOH1996 suppresses tumor growth without discernable toxicity at more than 10 time the compound's effective dose. In addition, AOH1996 works synergistically with known DNA damage agents to kill cancer cells. These results reveal TRC as cancer-selective vulnerability and demonstrated the therapeutic potential of AOH1996. Citation Format: Long Gu, Caroline Li, Robert Lingeman, Robert J. Hickey, Linda H. Malkas. Pharmacological targeting of transcription-replication conflict leads to anti-cancer efficacy with minimal side effects in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1269.

Abstract 2046: Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation

Abstract BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA. METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RNA FISH was used to measure mRNA export. RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown leads to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype. In addition, immunoprecipitation in cells and in vitro showed direct interaction between FANCD2 and SRSF1. We found that FANCD2 and SRSF1 knockdown leads to increased R-loops visualized by S9.6 immunofluorescence. Besides, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots. Additionally, we found that SRSF1 interacts with the mRNA export factor NXF1 in a FANCD2-dependent fashion and through RNA. Interestingly, SRSF1-NXF1 interaction is enhanced upon DNA damage. Finally, we found that FANCD2-deficient cells present defects in mRNA export, suggesting that SRSF1 and FANCD2 could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus. CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability. Citation Format: Anne Olazabal-Herrero, Allison M. Green, Xiaoyong Chen, Patrick Sung, Pillai M. Manoj, Gary M. Kupfer. Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2046.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

A novel role for Dun1 in the regulation of origin firing upon hyper-acetylation of H3K56.

During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader.

Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq.

R-loops are non-canonical nucleic structures composed of an RNA-DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. Here, we provide a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization. Graphic abstract: Figure 1.Overview of the strand-specific DRIP-seq protocol.

Age-associated deficient recruitment of 53BP1 in G1 cells directs DNA double-strand break repair to BRCA1/CtIP-mediated DNA-end resection.

DNA repair mechanisms play a crucial role in maintaining genome integrity. However, the increased frequency of DNA double-strand breaks (DSBs) and genome rearrangements in aged individuals suggests an age-associated DNA repair deficiency. Previous work from our group revealed a delayed firing of the DNA damage response in human mammary epithelial cells (HMECs) from aged donors. We now report a decreased activity of the main DSB repair pathways, the canonical non-homologous end-joining (c-NHEJ) and the homologous recombination (HR) in these HMECs from older individuals. We describe here a deficient recruitment of 53BP1 to DSB sites in G1 cells, probably influenced by an altered epigenetic regulation. 53BP1 absence at some DSBs is responsible for the age-associated DNA repair defect, as it permits the ectopic formation of BRCA1 foci while still in the G1 phase. CtIP and RPA foci are also formed in G1 cells from aged donors, but RAD51 is not recruited, thus indicating that extensive DNA-end resection occurs in these breaks although HR is not triggered. These results suggest an age-associated switch of DSB repair from canonical to highly mutagenic alternative mechanisms that promote the formation of genome rearrangements, a source of genome instability that might contribute to the aging process.