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The Journal of Biological Chemistry Nov 2023With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA-DNA hybrid and a displaced single-strand (ss) DNA,... (Review)
Review
With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA-DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA-DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA-DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases.
Topics: DNA; DNA Repair; R-Loop Structures; RNA; Humans; Animals; DNA-Binding Proteins; RNA-Binding Proteins
PubMed: 37778731
DOI: 10.1016/j.jbc.2023.105307 -
PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.Cell Feb 2024DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that...
DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Poly (ADP-Ribose) Polymerase-1; Humans
PubMed: 38320550
DOI: 10.1016/j.cell.2024.01.015 -
Trends in Biotechnology Aug 2023Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will... (Review)
Review
Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic and diagnostic landscape. However, CRISPR-Cas9, which edits DNA by activating DNA double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation repair is required. Prime editing, the latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without the requirement for DSBs. However, prime editing is still in its infancy, and further development is needed to improve editing efficiency and delivery strategies for therapeutic applications. We summarize latest developments in the optimization of prime editor (PE) variants with improved editing efficiency and precision. Moreover, we highlight some potential therapeutic applications.
Topics: CRISPR-Cas Systems; CRISPR-Associated Protein 9; Gene Editing; DNA Repair; DNA
PubMed: 37002157
DOI: 10.1016/j.tibtech.2023.03.004 -
Annual Review of Biochemistry Jun 2023Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main... (Review)
Review
Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.
Topics: Humans; Cockayne Syndrome; DNA Repair Enzymes; Transcription, Genetic; DNA Repair; DNA Damage; DNA; Carrier Proteins
PubMed: 37040775
DOI: 10.1146/annurev-biochem-052621-091205 -
Cell Sep 2023Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the...
Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.
Topics: Animals; Humans; Mice; Alzheimer Disease; DNA; DNA Breaks, Double-Stranded; DNA Repair; Neurodegenerative Diseases; Neurons; Single-Cell Analysis; Sequence Analysis, RNA; Genomic Instability
PubMed: 37774679
DOI: 10.1016/j.cell.2023.08.038 -
International Journal of Molecular... Nov 2023The intricate interplay between DNA damage response (DDR) and metabolism unveils a profound insight into the fundamental mechanisms governing the maintenance of genomic...
The intricate interplay between DNA damage response (DDR) and metabolism unveils a profound insight into the fundamental mechanisms governing the maintenance of genomic integrity [...].
Topics: Humans; DNA Repair; DNA Damage; Neoplasms
PubMed: 38003620
DOI: 10.3390/ijms242216430 -
Nature Sep 2023Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations. Paradoxically, the types of DNA rearrangements that are...
Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.
Topics: Humans; BRCA1 Protein; BRCA2 Protein; Chromosome Inversion; DNA Repair; Neoplasms; Translocation, Genetic; Homologous Recombination; Cytogenetic Analysis; Chromosome Aberrations
PubMed: 37587346
DOI: 10.1038/s41586-023-06461-2 -
Molecular Cell Aug 2023To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human...
To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.
Topics: Humans; CRISPR-Cas Systems; DNA Damage; Mutation; DNA Repair; Phenotype
PubMed: 37478847
DOI: 10.1016/j.molcel.2023.06.025 -
Nucleic Acids Research Sep 2023Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA...
Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.
Topics: Humans; Cell Line, Tumor; DNA Damage; DNA Repair; Histone Deacetylase 6; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 37503842
DOI: 10.1093/nar/gkad631 -
International Journal of Molecular... Nov 2023Following our first Special Issue, we are pleased to present this Special Issue in the , titled 'DNA Damage, DNA Repair, and Cancer: Second Edition' [...].
Following our first Special Issue, we are pleased to present this Special Issue in the , titled 'DNA Damage, DNA Repair, and Cancer: Second Edition' [...].
Topics: Humans; DNA Repair; DNA Damage; Neoplasms
PubMed: 38069158
DOI: 10.3390/ijms242316835