DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-helikasy metabolismus genetika MeSH
- DNA * metabolismus chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- fosforylace MeSH
- homeostáza genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proteinfosfatasa 2 metabolismus genetika MeSH
- R-smyčka MeSH
- RNA-helikasy metabolismus genetika MeSH
- RNA-polymerasa II * metabolismus MeSH
- RNA * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.
- MeSH
- crossing over (genetika) * MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- endonukleasy * metabolismus genetika MeSH
- homologní rekombinace MeSH
- resolvasy Hollidayova spojení metabolismus genetika MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus genetika MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.
- MeSH
- alternativní sestřih MeSH
- beta-katenin * metabolismus genetika MeSH
- fosforylace MeSH
- GSK3B metabolismus genetika MeSH
- HEK293 buňky MeSH
- kasein kinasa Ialfa * metabolismus genetika MeSH
- kinasa 3 glykogensynthasy metabolismus genetika MeSH
- lidé MeSH
- protein Wnt3A metabolismus genetika MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.
- MeSH
- chřipka lidská virologie MeSH
- elektronová kryomikroskopie * MeSH
- fosforylace MeSH
- genetická transkripce MeSH
- lidé MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- replikace viru MeSH
- RNA virová metabolismus genetika MeSH
- RNA-dependentní RNA-polymerasa * metabolismus chemie MeSH
- RNA-polymerasa II * metabolismus chemie MeSH
- vazba proteinů MeSH
- virové proteiny * metabolismus chemie genetika MeSH
- virus chřipky A * metabolismus genetika enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.
Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.
- MeSH
- DNA-helikasy * genetika metabolismus MeSH
- genetická transkripce MeSH
- lidé MeSH
- multifunkční enzymy genetika metabolismus MeSH
- neurodegenerativní nemoci MeSH
- R-smyčka MeSH
- regulace genové exprese MeSH
- RNA-helikasy * metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- terminace genetické transkripce * MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
- MeSH
- mikro RNA * genetika metabolismus MeSH
- myši MeSH
- ribonukleasa III * metabolismus MeSH
- RNA interference MeSH
- savci metabolismus MeSH
- transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
- MeSH
- buněčné linie MeSH
- fosforylace MeSH
- genetická transkripce MeSH
- genový knockdown MeSH
- lidé MeSH
- myši knockoutované MeSH
- neurony chemie metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- proteinové domény MeSH
- regulace genové exprese MeSH
- RNA-polymerasa II chemie genetika metabolismus MeSH
- RNA * chemie genetika metabolismus MeSH
- stabilita RNA MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1-RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin.
- MeSH
- chromatin metabolismus MeSH
- DNA-helikasy genetika MeSH
- DNA biosyntéza MeSH
- methylmethansulfonát toxicita MeSH
- mutace MeSH
- nestabilita genomu * MeSH
- poškození DNA * MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- rekombinace genetická MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- strukturní homologie proteinů MeSH
- suprese genetická MeSH
- transportní proteiny chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.
- MeSH
- "flap" endonukleasy genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- endonukleasy genetika metabolismus MeSH
- proliferační antigen buněčného jádra genetika metabolismus MeSH
- rekombinace genetická MeSH
- replikace DNA MeSH
- replikační protein C genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
