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Molecular Cell Jan 2024Completion of DNA replication relies on the ability of replication forks to traverse various types of DNA damage, actively transcribed regions, and structured DNA. The...
Completion of DNA replication relies on the ability of replication forks to traverse various types of DNA damage, actively transcribed regions, and structured DNA. The mechanisms enabling these processes are here referred to as DNA damage tolerance pathways. Here, we depict the stalled DNA replication fork structures with main DNA transactions and key factors contributing to the bypass of such blocks, replication restart, and completion. To view this SnapShot, open or download the PDF.
Topics: DNA Damage; DNA Damage Tolerance; DNA
PubMed: 38181760
DOI: 10.1016/j.molcel.2023.11.031 -
Nature Communications Dec 2023DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously...
DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.
Topics: Humans; DNA Replication; DNA-Directed DNA Polymerase; DNA; Exonucleases
PubMed: 38151585
DOI: 10.1038/s41467-023-44198-8 -
Chromosoma Jan 2024Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases... (Review)
Review
Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time.
Topics: Humans; Telomere Homeostasis; DNA Replication; DNA Repair; DNA; DNA Damage; Telomere
PubMed: 37581649
DOI: 10.1007/s00412-023-00807-5 -
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 -
Nature Nov 2023In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements...
In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements leads to genome instability and human diseases, including cancer and ageing. Although the role of H3K9me3 in the establishment and maintenance of heterochromatin silencing has been extensively studied, the pattern and mechanism that underlie the partitioning of parental H3K9me3 at replicating DNA strands are unknown. Here we report that H3K9me3 is preferentially transferred onto the leading strands of replication forks, which occurs predominantly at long interspersed nuclear element (LINE) retrotransposons (also known as LINE-1s or L1s) that are theoretically transcribed in the head-on direction with replication fork movement. Mechanistically, the human silencing hub (HUSH) complex interacts with the leading-strand DNA polymerase Pol ε and contributes to the asymmetric segregation of H3K9me3. Cells deficient in Pol ε subunits (POLE3 and POLE4) or the HUSH complex (MPP8 and TASOR) show compromised H3K9me3 asymmetry and increased LINE expression. Similar results were obtained in cells expressing a MPP8 mutant defective in H3K9me3 binding and in TASOR mutants with reduced interactions with Pol ε. These results reveal an unexpected mechanism whereby the HUSH complex functions with Pol ε to promote asymmetric H3K9me3 distribution at head-on LINEs to suppress their expression in S phase.
Topics: Humans; DNA Replication; Gene Silencing; Histones; Long Interspersed Nucleotide Elements; Lysine; Methylation; S Phase
PubMed: 37938774
DOI: 10.1038/s41586-023-06711-3 -
Current Opinion in Structural Biology Aug 2023The replication machinery frequently encounters DNA damage and other structural impediments that inhibit progression of the replication fork. Replication-coupled... (Review)
Review
The replication machinery frequently encounters DNA damage and other structural impediments that inhibit progression of the replication fork. Replication-coupled processes that remove or bypass the barrier and restart stalled forks are essential for completion of replication and for maintenance of genome stability. Errors in replication-repair pathways lead to mutations and aberrant genetic rearrangements and are associated with human diseases. This review highlights recent structures of enzymes involved in three replication-repair pathways: translesion synthesis, template switching and fork reversal, and interstrand crosslink repair.
Topics: Humans; DNA Damage; DNA Repair; DNA Replication; Mutation; Genomic Instability
PubMed: 37269798
DOI: 10.1016/j.sbi.2023.102618 -
Current Opinion in Structural Biology Aug 2023Eukaryotic DNA replication is performed by the replisome, a large and dynamic multi-protein machine endowed with the required enzymatic components for the synthesis of... (Review)
Review
Eukaryotic DNA replication is performed by the replisome, a large and dynamic multi-protein machine endowed with the required enzymatic components for the synthesis of new DNA. Recent cryo-electron microscopy (cryoEM) analyses have revealed the conserved architecture of the core eukaryotic replisome, comprising the CMG (Cdc45-MCM-GINS) DNA helicase, the leading-strand DNA polymerase epsilon, the Timeless-Tipin heterodimer, the hub protein AND-1 and the checkpoint protein Claspin. These results bid well for arriving soon at an integrated understanding of the structural basis of semi-discontinuous DNA replication. They further set the scene for the characterisation of the mechanisms that interface DNA synthesis with concurrent processes such as DNA repair, propagation of chromatin structure and establishment of sister chromatid cohesion.
Topics: DNA Helicases; Cryoelectron Microscopy; Saccharomyces cerevisiae Proteins; DNA Replication; DNA; Eukaryota; Cell Cycle Proteins
PubMed: 37244171
DOI: 10.1016/j.sbi.2023.102612 -
Journal of Virology Feb 2024Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are... (Review)
Review
Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.
Topics: Humans; Adenoviridae; Adenoviruses, Human; DNA Replication; DNA, Viral; DNA-Binding Proteins; Viral Proteins; Virus Replication
PubMed: 38197632
DOI: 10.1128/jvi.01885-23 -
Science (New York, N.Y.) Mar 2024DNA replication is initiated at multiple loci to ensure timely duplication of eukaryotic genomes. Sister replication forks progress bidirectionally, and replication...
DNA replication is initiated at multiple loci to ensure timely duplication of eukaryotic genomes. Sister replication forks progress bidirectionally, and replication terminates when two convergent forks encounter one another. To investigate the coordination of replication forks, we developed a replication-associated in situ HiC method to capture chromatin interactions involving nascent DNA. We identify more than 2000 fountain-like structures of chromatin contacts in human and mouse genomes, indicative of coupling of DNA replication forks. Replication fork interaction not only occurs between sister forks but also involves forks from two distinct origins to predetermine replication termination. Termination-associated chromatin fountains are sensitive to replication stress and lead to coupled forks-associated genomic deletions in cancers. These findings reveal the spatial organization of DNA replication forks within the chromatin context.
Topics: Animals; Humans; Mice; Chromatin; DNA; DNA Replication; Genome, Human; Protein Conformation; High-Throughput Nucleotide Sequencing
PubMed: 38484065
DOI: 10.1126/science.adj7606 -
International Journal of Molecular... Nov 2023Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is... (Review)
Review
Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.
Topics: Humans; DNA Replication; DNA Repair; DNA Damage; Endonucleases; Genomic Instability; DNA; Nucleotides
PubMed: 38069223
DOI: 10.3390/ijms242316903