stalling
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Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSβ, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSβ, MutLβ, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.
- MeSH
- DNA-helikasy genetika metabolismus MeSH
- DNA genetika MeSH
- lidé MeSH
- proteiny FANC * genetika metabolismus MeSH
- R-smyčka * MeSH
- replikace DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.
- MeSH
- aktivace lymfocytů MeSH
- Burkittův lymfom genetika imunologie MeSH
- cytidindeaminasa genetika MeSH
- genetická transkripce MeSH
- imunoglobuliny genetika metabolismus MeSH
- kur domácí MeSH
- lidé MeSH
- mutace genetika MeSH
- mutageneze cílená MeSH
- podskupiny B-lymfocytů imunologie MeSH
- ptačí proteiny genetika metabolismus MeSH
- RNA-polymerasa II genetika metabolismus MeSH
- rozmanitost protilátek MeSH
- somatická hypermutace imunoglobulinových genů MeSH
- zesilovače transkripce genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.
- MeSH
- DNA opravný a rekombinační protein Rad52 metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-ligasy metabolismus MeSH
- DNA-polymerasa III metabolismus MeSH
- endodeoxyribonukleasy metabolismus MeSH
- endonukleasy genetika metabolismus MeSH
- genetická transkripce genetika MeSH
- HeLa buňky MeSH
- helikasy RecQ metabolismus fyziologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- R-smyčka genetika fyziologie MeSH
- rekombinasa Rad51 genetika metabolismus fyziologie MeSH
- replikace DNA genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
- MeSH
- elektronová kryomikroskopie MeSH
- elongační faktory chemie genetika metabolismus ultrastruktura MeSH
- Escherichia coli genetika metabolismus MeSH
- iniciační faktory chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- messenger RNA chemie genetika metabolismus MeSH
- mutace MeSH
- peptidy chemie metabolismus MeSH
- proteiny vázající RNA chemie metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus ultrastruktura MeSH
- proteosyntéza MeSH
- ribozomy chemie metabolismus ultrastruktura MeSH
- RNA transferová chemie genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely present in the translation and RNA metabolism protein complexes. Biochemical analysis validates OTUD1 association with ribosome subunits, elongation factors and the E3 ubiquitin ligase ZNF598 but not with the translation initiation machinery. OTUD1 catalytic activity suppresses polyA triggered ribosome stalling through inhibition of ZNF598-mediated RPS10 ubiquitination and stimulates formation of polysomes. Finally, analysis of gene expression suggests that OTUD1 regulates the stability of rare codon rich mRNAs by antagonizing ZNF598.
Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.
- MeSH
- biokatalýza MeSH
- biologické modely MeSH
- dimerizace MeSH
- konformace proteinů MeSH
- messenger RNA genetika metabolismus ultrastruktura MeSH
- podjednotky ribozomu metabolismus MeSH
- proteiny z Escherichia coli chemie metabolismus ultrastruktura MeSH
- ribozomy metabolismus ultrastruktura MeSH
- stabilita RNA MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.
- MeSH
- Bacillus subtilis enzymologie genetika MeSH
- bakteriální proteiny metabolismus MeSH
- bakteriální RNA genetika metabolismus MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- exoribonukleasy metabolismus MeSH
- genetická transkripce MeSH
- messenger RNA genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
2., verb. und erweit. Aufl. x, 144 s. : il. ; 24 cm
- Konspekt
- Fyzioterapie. Psychoterapie. Alternativní lékařství
- NLK Obory
- terapie
- NLK Publikační typ
- studie
- MeSH
- nemocnice - restrukturalizace MeSH
- obložnost MeSH
- reforma zdravotní péče MeSH
- Geografické názvy
- Maďarsko MeSH