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 spoje metabolismus   genetika   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• endonukleasy * MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• resolvasy Hollidayova spoje MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Yen1 protein, S cerevisiae MeSH Prohlížeč

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
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• jednovláknová DNA MeSH
• nukleotidy MeSH
• proteiny Caenorhabditis elegans MeSH
• rekombinasa Rad51 MeSH

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We concluded that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.

• Klíčová slova
• DNA repair, Schizosaccharomyces pombe, Sfr1, Swi5, meiosis, phosphorylation, recombination,
• MeSH
• fosforylace MeSH
• homologní rekombinace * MeSH
• meióza MeSH
• oprava DNA * MeSH
• poškození DNA * MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Schizosaccharomyces pombe - proteiny MeSH
• Sfr1 protein, S pombe MeSH Prohlížeč
• Swi5 protein, S pombe MeSH Prohlížeč

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
• Názvy látek
• adenosintrifosfát MeSH
• helikasy RecQ MeSH
• jednovláknová DNA MeSH
• molekulární motory MeSH
• RAD51 protein, human MeSH Prohlížeč
• RECQL5 protein, human MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• RPA1 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• DNA damage tolerance, double-strand break repair, genomic instability, homologous recombination, replication fork 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
• Názvy látek
• exodeoxyribonukleasy MeSH
• fosfoproteiny MeSH
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• TREX2 protein, human MeSH Prohlížeč

RECQ5 belongs to the RecQ family of DNA helicases. It is conserved from Drosophila to humans and its deficiency results in genomic instability and cancer susceptibility in mice. Human RECQ5 is known for its ability to regulate homologous recombination by disrupting RAD51 nucleoprotein filaments. It also binds to RNA polymerase II (RNAPII) and negatively regulates transcript elongation by RNAPII. Here, we summarize recent studies implicating RECQ5 in the prevention and resolution of transcription-replication conflicts, a major intrinsic source of genomic instability during cancer development.

• Klíčová slova
• DNA repair, R-loops, RECQ5, genomic instability, replication stress, transcription-replication conflicts,
• MeSH
• DNA genetika   metabolismus   MeSH
• genetická transkripce genetika   MeSH
• helikasy RecQ genetika   metabolismus   fyziologie   MeSH
• lidé MeSH
• nestabilita genomu MeSH
• replikace DNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• helikasy RecQ MeSH

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

• MeSH
• adeninnukleotidy metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• biologická evoluce MeSH
• elektronová kryomikroskopie MeSH
• jednovláknová DNA metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• rekombinasa Rad51 genetika   metabolismus   ultrastruktura   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adeninnukleotidy MeSH
• adenosintrifosfát MeSH
• jednovláknová DNA MeSH
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.

• MeSH
• chromatidy chemie   metabolismus   MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• jaderné proteiny chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• malé modifikační proteiny související s ubikvitinem chemie   genetika   metabolismus   MeSH
• nestabilita genomu MeSH
• poškození DNA MeSH
• proteinové domény MeSH
• proteiny buněčného cyklu MeSH
• regulace genové exprese u hub * MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• replikace DNA MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA fungální MeSH
• DNA vazebné proteiny MeSH
• endonukleasy MeSH
• Esc2 protein, S cerevisiae MeSH Prohlížeč
• jaderné proteiny MeSH
• křížová struktura DNA MeSH
• malé modifikační proteiny související s ubikvitinem MeSH
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

• Klíčová slova
• DNA repair, Rad51, Rad51 paralogs, filaments, genome stability, homologous recombination,
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• intermediární filamenta genetika   metabolismus   MeSH
• jednovláknová DNA genetika   MeSH
• multiproteinové komplexy metabolismus   MeSH
• proteiny Caenorhabditis elegans genetika   metabolismus   MeSH
• rekombinační oprava DNA MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• jednovláknová DNA MeSH
• multiproteinové komplexy MeSH
• proteiny Caenorhabditis elegans MeSH
• rad-51 protein, C elegans MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• RFS-1 protein, C elegans MeSH Prohlížeč
• RIP-1 protein, C elegans MeSH Prohlížeč
• transportní proteiny MeSH

Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Higher BRCA1 positivity is associated with shorter survival of glioma patients and the abrogation of BRCA1 function in GBM enhances RS, DNA damage (DD) accumulation and impairs tumour growth. Mechanistically, we identify a novel role of BRCA1 as a transcriptional co-activator of RRM2 (catalytic subunit of ribonucleotide reductase), whereby BRCA1-mediated RRM2 expression protects GBM cells from endogenous RS, DD and apoptosis. Notably, we show that treatment with a RRM2 inhibitor triapine reproduces the BRCA1-depletion GBM-repressive phenotypes and sensitizes GBM cells to PARP inhibition. We propose that GBM cells are addicted to the RS-protective role of the BRCA1-RRM2 axis, targeting of which may represent a novel paradigm for therapeutic intervention in GBM.

• MeSH
• analýza přežití MeSH
• glioblastom genetika   metabolismus   patologie   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory mozku genetika   metabolismus   patologie   MeSH
• protein BRCA1 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů * MeSH
• replikace DNA genetika   MeSH
• retrospektivní studie MeSH
• ribonukleosiddifosfátreduktasa genetika   metabolismus   MeSH
• RNA interference MeSH
• transplantace heterologní MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BRCA1 protein, human MeSH Prohlížeč
• protein BRCA1 MeSH
• ribonucleotide reductase M2 MeSH Prohlížeč
• ribonukleosiddifosfátreduktasa MeSH