Jul 2026· Proceedings of the National Academy of Sciences of the United States of America· Vol 123 28, pp.
e2529943123
· 0 citations· 56 references
Medicine
TL;DR
It is demonstrated that defective single-strand break (SSB) repair acts as a potent trigger for genome-wide TE derepression and extensive de novo DNA methylation in Arabidopsis.
Abstract
Plant genomic and epigenomic integrity are perpetually threatened by exogenous and endogenous DNA damage. However, the interplay between DNA damage, DNA methylation (5mC), and transposable element (TE) activity remains poorly understood. Here, we demonstrate that defective single-strand break (SSB) repair acts as a potent trigger for genome-wide TE derepression and extensive de novo DNA methylation in Arabidopsis. Mutations in ZDP/APE2, which encode conserved DNA 3'-end repair enzymes, impair the repair of 3'-blocked SSBs arising from base excision repair, ultimately leading to widespread TE activation. Concurrently, inefficient SSB repair activates the ATR-SOG1-mediated DNA damage response, which enhances the RNA-directed DNA methylation (RdDM) pathway to counteract TE activation by depositing 5mC. Paradoxically, the resulting methylation is excised by the DNA demethylase ROS1-a process that itself generates 3'-blocked SSBs requiring resolution by ZDP/APE2. In zdp ape2 mutants, ROS1-mediated 5mC excision produces additional SSBs, which in turn reactivate RdDM. This establishes a self-sustaining SSB-5mC cycle that perpetuates DNA damage and drives massive TE activation in the mutant. Our findings reveal a critical mechanistic link between SSB repair, DNA methylation dynamics, and TE derepression, positioning defective SSB repair as a major inducer of epigenomic instability.
Abstract DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions because they disrupt the connectivity of the DNA strand. Homologous recombination (HR) is a high-fidelity DSB repair pathway that copies the sequence spanning the break from a homologous template, but how DNA ends are held together during HR remains unclear. Here we demonstrate that the proline-serine-threonine (PST) repeat region of Mediator of DNA Damage Checkpoint 1 (MDC1) is a multivalent nucleosome-binding domain, sufficient to tether chromatin in multiple contexts. In interphase, the constitutive chromatin association of MDC1 is critical for RAD51 loading and efficient HR. In mitosis, PST-mediated chromatin binding is attenuated by phosphorylation, preventing aberrant chromosomal interactions while preserving DNA tethering by the MDC1–TOPBP1–CIP2A complex. In total, this work demonstrates that the PST repeat region of MDC1 is a multivalent nucleosome-binding domain with tunable affinity that supports DSB repair by HR and maintains genome stability during mitosis.
Joshua R. Heyza, Mariia Mikhova, Cody Phillips et al.· Nucleic Acids Research· 0 citations
Oxidative DNA damage is a common threat to genomic integrity, arising from endogenous metabolic processes and environmental exposures. If unrepaired, such oxidative DNA damage promotes mutagenesis and genomic instability. Cells counter this through base excision repair (BER), a multi-step pathway requiring the coordinated action of several proteins. Central to BER, DNA polymerase beta (pol ꞵ) locates single-nucleotide (1-nt) gaps and inserts the correct nucleotide, while x-ray repair cross-complementing 1 (XRCC1) is a scaffold protein that forms a stable complex with pol ꞵ to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol β is proposed to be recruited to DNA damage by undamaged DNA scanning interactions, but this behavior has not yet been directly observed. Additionally, the influence of other BER proteins on pol ꞵ recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol ꞵ and XRCC1. We characterize each factor individually, examine their behavior as the pol ꞵ-XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol ꞵ in BER. We find that pol ꞵ locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol β search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol ꞵ are present, the pol ꞵ-1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol ꞵ. Together, these findings demonstrate that XRCC1 reshapes pol β search behavior to promote efficient local damage recognition, providing a mechanistic basis for how BER factors coordinate lesion detection and processing to maintain genomic stability. Significance Statement DNA repair proteins must locate rare sites of damage hidden within millions of undamaged bases. Using single-molecule imaging with optical tweezers, we directly visualized how DNA polymerase ꞵ and its scaffold partner XRCC1 search for and engage DNA damage. Alone, pol β finds damage exclusively through 3D collisions, whereas XRCC1 scans along DNA by 1D hopping. When the two proteins form a complex, XRCC1 confers its scanning ability on pol β, expanding the search strategies available for damage detection. These findings reveal a mechanism by which scaffold proteins remodel the damage search process of their partners, providing insight into how base excision repair is coordinated to maintain genome stability.
Spencer H. Thompson, Kaitlin M. DeHart, M. Schaich et al.· bioRxiv· 0 citations
The DNA damage response preserves genome integrity by detecting DNA lesions, activating checkpoint signalling, and coordinating repair with cell-cycle control. Defective or incomplete repair can promote mutation accumulation, chromosomal instability, cancer development, and ageing-associated diseases. In this review, we discuss the human single-stranded DNA-binding proteins hSSB1 and hSSB2, with an emphasis on their roles in ssDNA-rich repair intermediates generated during double-strand break repair, replication stress, oxidative base damage, telomere maintenance, and selected ultraviolet-damage responses. Both proteins contain oligonucleotide/oligosaccharide-binding fold domains that support ssDNA recognition and provide platforms for protein–protein interactions within DNA repair and chromatin-associated pathways. Current evidence identifies hSSB1 as a major regulator of ataxia telangiectasia mutated (ATM)/MRE11–RAD50–NBS1 (MRN)-dependent double-strand break signalling, RAD51-associated homologous recombination, human 8-oxoguanine DNA glycosylase 1 (hOGG1)-mediated repair of 8-oxo-guanine, replication-fork stability, and telomere protection. By contrast, hSSB2 remains less extensively characterised and appears to act in more restricted or context-dependent settings, including the cellular response to ultraviolet-induced DNA damage. We also discuss how post-translational modifications, SOSS/Integrator-associated complexes, transcriptional regulation, and possible epigenetic mechanisms shape hSSB1 and hSSB2 function. Finally, we highlight unresolved questions concerning the extent of functional overlap between these paralogues, the lack of damage-context-specific genome-wide binding maps, and the need to validate whether altered hSSB1 or hSSB2 expression can be exploited as a biomarker or therapeutic vulnerability in cancer.
Armin Sharifi, Amila Suraweera, K. J. O'Byrne et al.· Cellular Oncology· 0 citations
Nucleotide excision repair (NER) is a crucial DNA repair pathway that is orchestrated by transcription factor IIH (TFIIH) in eukaryotic cells. TFIIH is a multifunctional complex that contains two DNA helicase/DNA translocase subunits and a kinase module, different subsets of which act in NER, transcription initiation, and cell cycle control. To ensure fidelity despite multifunctionality, the DNA helicase activity of TFIIH is autoinhibited in its free form or when the factor engages in transcription initiation. While the release of the kinase module has been identified as a key step in TFIIH activation, the molecular mechanisms controlling this step and concomitant structural changes in TFIIH are incompletely understood. Here, we determine high-resolution structures of three NER intermediates that visualize how TFIIH arrives at sites of DNA damage in an autoinhibited state and how autoinhibition is released via previously undescribed intermediates. These findings contribute to a mechanistic understanding of human DNA repair.
Natàlia de Martín Garrido, Callum A. F. Haste, Junjie Feng et al.· Science Advances· 0 citations
Cells undergoing division mount a unique response to DNA damage that ensures accurate chromosome segregation. The CIP2A-TOPBP1 complex has emerged as an important mitotic genome maintenance factor, but its function remains unclear. Here, we report that DDIAS is a DNA-binding effector of the CIP2A pathway in human cells. DDIAS physically interacts with TOPBP1, and its inactivation causes synthetic lethality with BRCA1 and BRCA2 deficiency. Homologous recombination (HR)-deficient tumors upregulate DDIAS to enable HR-deficient cells to tolerate their genomic instability. Mechanistically, DDIAS is a single-stranded DNA (ssDNA)-binding protein that promotes the repair of ssDNA carried from interphase into mitosis. Mitotic ssDNA in HR-deficient cells is exacerbated by poly(ADP-ribose) polymerase (PARP) inhibition, and DDIAS-dependent suppression of these lesions involves mitotic DNA synthesis, which promotes accurate chromosome segregation and survival. We propose that DDIAS defines a mitotic DNA repair system downstream of CIP2A that mitigates the threat of mitotic ssDNA for genome integrity.
Yibo Xue, Faisal Bin Rashed, Daniel Y. L. Mao et al.· Molecules and Cells· 0 citations
DNA double-strand breaks, including those arising from DNA interstrand crosslinks, are highly cytotoxic forms of DNA damage. Their precise repair by homologous recombination (HR) is essential for maintaining genomic stability. During HR, timely disassembly of the core machinery, RAD51 nucleoprotein filaments, is critical. The FIGNL1-FIRRM (also known as C1orf112) AAA+ ATPase has recently been identified by us and others as a novel anti-recombinase that facilitates RAD51 disassembly via an ATPase-driven unfolding mechanism distinct from all previously characterized pathways. In this study, we identify MACIR/C5orf30 as an adaptor for FIGNL1-FIRRM unfoldase. MACIR directly binds FIRRM, forming a ternary complex that promotes RAD51 filament disassembly and facilitates repair of DNA damage induced by agents including aldehydes and platinum drugs. MACIR loss leads to DNA damage accumulation and genome instability, activates inflammatory signaling, linking MACIR deficiency to autoimmune and tumor-related pathologies. We further show that MACIR's direct interactions with FIRRM and DNA are both required for DNA repair, indicating that MACIR functionally links these components. High expression of MACIR-FIRRM-FIGNL1 correlates with poor prognosis in ovarian cancer patients, and pharmacological targeting of this complex with the inhibitors (small-molecule compound 8 and 10) we identified sensitizes ovarian cancer cells to platinum drugs. Therefore, our findings reveal MACIR as a critical regulatory adaptor within this RAD51-unfolding complex, which functions in an additional layer of control over DNA repair and genome stability, and suggest that its inhibition could enhance chemotherapeutic efficacy in cancer treatment.
Tao Zhou, Ming Pang, Xinxin Liang et al.· Proceedings of the National...· 0 citations
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