Biology subjects

Sebesta, M.

Publications and source records attributed to Sebesta, M..

6 recordsLinked to original sources

bioRxiv2024

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks

DNA double strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death and 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 trimeric SOSS1 (comprising INTS3, INIP, and hSSB1) to form a tetrameric SOSS1 complex following DNA damage. INTS6 binds to DNA:RNA hybrids and plays a crucial role in Protein Phosphatase 2 (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-induced 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 SOSS1 complex in the autoregulation of DNA:RNA dynamics and the promotion of efficient DNA repair.

molecular biology↗

bioRxiv2023

Structural and functional characterisation of the interaction between the influenza A virus RNA polymerase and the CTD of host RNA Polymerase II

Influenza A viruses (IAV), 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 IAV polymerase bound to a synthetic pS5 CTD heptad repeat peptide. 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 mediating 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 with normal replication, while other mutants display defects in both transcription and 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.

microbiology↗

bioRxiv2023

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

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/recombination intermediates. Here, we employed a biochemical approach to demonstrate 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 confirm 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.

molecular biology↗

bioRxiv2023

Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

The most toxic forms of DNA damage are double-strand breaks (DSBs). We have previously shown that RNA polymerase II (RNAPII), phosphorylated at tyrosine 1 (Y1P) on the C- terminal domain, transcribes RNA at DSBs to promote efficient DNA repair. However, it is still unknown how transcription is regulated at DSBs. Here, we show that the trimeric SOSS1 complex (hSSB1, INTS3, and c9orf80) binds to Y1P RNAPII in response to DNA damage, hSSB1 binds to R-loops, and formation of the SOSS1 complex is required for the coexistence of replication protein A (RPA) and hSSB1 at DSBs. The damage-activated tyrosine kinase c- Abl phosphorylates hSSB1 to enable its binding to Y1P RNAPII and its recruitment to DSBs. Finally, we show both in vitro and in vivo that the SOSS1 complex and RNAPII form dynamic repair compartments at DSBs via liquid-liquid phase separation (LLPS). The loss of the trimeric SOSS1 leads to impaired DNA repair, highlighting its biological importance in the RNA-dependent DNA damage response. TeaserTrimeric SOSS1 complex and transcription contribute to phase separation at double-strand DNA breaks.

molecular biology↗

bioRxiv2022

Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor

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.

biochemistry↗

bioRxiv2022

Molecular basis of indispensable accuracy of mammalian miRNA biogenesis

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicers highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicers DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural "mold" for adaptive miRNA evolution.

molecular biology↗