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Proceedings of the National Academy of... Sep 2023Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric...
Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.
Topics: Humans; Centromere; Chromosome Aberrations; Cytogenetics; DNA Replication; Genomic Instability; Isochromosomes
PubMed: 37722039
DOI: 10.1073/pnas.2303752120 -
Nature Communications Aug 2023Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication...
Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We conclude that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.
Topics: Humans; S Phase; Cell Division; Cell Proliferation; Cell Cycle; Cell Survival; Ubiquitin-Protein Ligases
PubMed: 37604812
DOI: 10.1038/s41467-023-40695-y -
Nature Structural & Molecular Biology May 2024The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and...
The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.
Topics: Telomere; ADP-Ribosylation; DNA Replication; Poly (ADP-Ribose) Polymerase-1; Humans; DNA; Animals; Mice; Adenosine Diphosphate Ribose; Genomic Instability; Telomere Shortening
PubMed: 38714889
DOI: 10.1038/s41594-024-01279-6 -
Nature Communications Jul 2023RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient...
RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5' RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.
Topics: Male; Humans; Prostatic Neoplasms; Rad51 Recombinase; Mutation; DNA Replication; Adenosine Triphosphate; DNA-Binding Proteins
PubMed: 37488098
DOI: 10.1038/s41467-023-40096-1 -
DNA Repair Oct 2023Guanine-rich DNA sequences can fold into stable four-stranded structures called G-quadruplexes or G4s. Research in the past decade demonstrated that G4 structures are... (Review)
Review
Guanine-rich DNA sequences can fold into stable four-stranded structures called G-quadruplexes or G4s. Research in the past decade demonstrated that G4 structures are widespread in the genome and prevalent in regulatory regions of actively transcribed genes. The formation of G4s has been tightly linked to important biological processes including regulation of gene expression and genome maintenance. However, they can also pose a serious threat to genome integrity especially by impeding DNA replication, and G4-associated somatic mutations have been found accumulated in the cancer genomes. Specialised DNA helicases and single stranded DNA binding proteins that can resolve G4 structures play a crucial role in preventing genome instability. The large variety of G4 unfolding proteins suggest the presence of multiple G4 resolution mechanisms in cells. Recently, there has been considerable progress in our detailed understanding of how G4s are resolved, especially during DNA replication. In this review, we first discuss the current knowledge of the genomic G4 landscapes and the impact of G4 structures on DNA replication and genome integrity. We then describe the recent progress on the mechanisms that resolve G4 structures and their physiological relevance. Finally, we discuss therapeutic opportunities to target G4 structures.
Topics: G-Quadruplexes; DNA Replication; DNA Helicases; DNA-Binding Proteins; Regulatory Sequences, Nucleic Acid
PubMed: 37572578
DOI: 10.1016/j.dnarep.2023.103552 -
The EMBO Journal Mar 2024Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and...
Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.
Topics: DNA; DNA Replication; DNA-Binding Proteins; MRE11 Homologue Protein; Rad51 Recombinase; Humans
PubMed: 38360996
DOI: 10.1038/s44318-024-00038-z -
EcoSal Plus Dec 2023DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process... (Review)
Review
DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process includes different phases leading to the physical separation of the replicons and their movement toward the future daughter cells. Here, we review these phases and processes in enterobacteria with emphasis on the molecular mechanisms at play and their controls.
Topics: Enterobacteriaceae; Chromosomes, Bacterial; DNA; Replicon; DNA Replication
PubMed: 37220081
DOI: 10.1128/ecosalplus.esp-0038-2020 -
Annual Review of Genetics Nov 2023Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA... (Review)
Review
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.
Topics: Animals; Transcription, Genetic; DNA Replication; Genomic Instability; Eukaryota; DNA Damage; Mammals
PubMed: 37552891
DOI: 10.1146/annurev-genet-080320-031523 -
ELife Mar 2024R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can...
R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.
Topics: Humans; ATPases Associated with Diverse Cellular Activities; DNA; DNA Damage; DNA Replication; DNA-Binding Proteins; Genomic Instability; Hydrolases; Zinc Fingers
PubMed: 38488661
DOI: 10.7554/eLife.89981 -
Cancer Science Jan 2024Epigenetic modifications are significant in tumor pathogenesis, wherein the process of histone demethylation is indispensable for regulating gene transcription,... (Review)
Review
Epigenetic modifications are significant in tumor pathogenesis, wherein the process of histone demethylation is indispensable for regulating gene transcription, apoptosis, DNA replication, and repair of damaged DNA. The lysine demethylases (KDMs) serve an essential role in the aforementioned processes, with particular emphasis on the KDM4 family, also referred to as JMJD2. Multiple studies have underscored the significance of the KDM4 family in the regulation of various biological processes including, but not limited to, the cell cycle, DNA repair mechanisms, signaling pathways, and the progression of tumor formation. Nevertheless, it is imperative to elucidate the underlying mechanism of KDM4B, which belongs to the KDM4 gene family. This review presents a comprehensive examination of the structure, mechanism, and function of KDM4B, as well as a critical analysis of the current body of research pertaining to its involvement in tumorigenesis and development. Furthermore, this review explores the potential therapeutic strategies that specifically target KDM4B.
Topics: Humans; Neoplasms; DNA Repair; Cell Cycle; Signal Transduction; DNA Replication; Jumonji Domain-Containing Histone Demethylases
PubMed: 37923555
DOI: 10.1111/cas.16005