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
BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.
- MeSH
- DNA vazebné proteiny * genetika metabolismus MeSH
- DNA-topoisomerasy I genetika metabolismus MeSH
- endonukleasy * genetika metabolismus MeSH
- fosfodiesterasy * genetika metabolismus MeSH
- oprava DNA MeSH
- poškození DNA MeSH
- Saccharomyces cerevisiae - proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
- MeSH
- adenosindifosfát farmakologie MeSH
- adenosintrifosfát farmakologie MeSH
- Caenorhabditis elegans metabolismus MeSH
- druhová specificita MeSH
- fluorescence MeSH
- interferometrie MeSH
- jednovláknová DNA metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- rekombinasa Rad51 chemie metabolismus MeSH
- sekvenční homologie aminokyselin * MeSH
- stabilita proteinů účinky léků MeSH
- vazba proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In vitro analysis of posttranslational modifications such as sumoylation provides a great tool to not only identify the target proteins but also to characterize the specific effects of this modification on the protein features and uncover possible regulatory mechanism. In this chapter, we will describe the purification of yeast SUMO machinery proteins and their use to identify SUMO modification of target proteins in vitro. Furthermore, we will show several examples characterizing the effect of sumoylation on the biochemical activities of various proteins involved in homologous recombination (HR) that helped to better understand the regulatory role of this modification.
- MeSH
- Escherichia coli genetika růst a vývoj metabolismus MeSH
- homologní rekombinace * MeSH
- komplexy ubikvitinligas metabolismus MeSH
- malé modifikační proteiny související s ubikvitinem metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- rekombinantní proteiny izolace a purifikace MeSH
- sumoylace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
- MeSH
- adenosintrifosfát metabolismus MeSH
- bodová mutace MeSH
- helikasy RecQ genetika metabolismus ultrastruktura MeSH
- homologní rekombinace * MeSH
- hydrolýza MeSH
- jednovláknová DNA metabolismus ultrastruktura MeSH
- kinetika MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- missense mutace MeSH
- molekulární motory metabolismus ultrastruktura MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- replikační protein A metabolismus MeSH
- substrátová specifita MeSH
- zobrazení jednotlivé molekuly * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.
- MeSH
- exodeoxyribonukleasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- myši MeSH
- rekombinasa Rad51 biosyntéza genetika metabolismus MeSH
- replikace DNA * MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.
- MeSH
- antibakteriální látky farmakologie MeSH
- Escherichia coli účinky léků genetika účinky záření MeSH
- koncentrace vodíkových iontů MeSH
- kyselina nalidixová farmakologie MeSH
- mikrobiální viabilita účinky léků účinky záření MeSH
- nestabilita genomu účinky léků genetika účinky záření MeSH
- poškození DNA účinky léků genetika účinky záření MeSH
- propidium farmakologie MeSH
- průtoková cytometrie MeSH
- retardační test MeSH
- rifampin farmakologie MeSH
- ultrafialové záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
