DNA double-strand breaks (DSBs) are formed in meiosis, so their repair in the homologous recombination (HR) pathway will lead to crossover formation, which is essential for successful chromosome segregation. HR contains 2 subpathways: synthesis-dependent strand annealing (SDSA) that creates noncrossover and double Holliday junction (dHJ) that generates crossovers. RAD-51 is a protein essential to the formation of all products of HR, as it assembles on the processed DSB, allowing the invasion of the single-stranded DNA into a region of homology. RAD-51 is removed by RAD-54.L after invasion to allow for repair to occur. Here, we investigate a separation of function allele of rad-51, rad-51::FLAG, as compared to 2 other RAD-51 alleles: rad-51::degron and GFP::rad-51. rad-51::FLAG displays slowed repair kinetics, resulting in an accumulation of RAD-51 foci. rad-51::FLAG worms also activate the DSB checkpoint, but to a less extant than that of rad-51 null mutants. In a proximity ligation assay, RAD-54.L and RAD-51 show enriched colocalization in rad-51::FLAG germlines (but not in rad-51::degron), consistent with stalling at the strand invasion step in HR. The defects in RAD-51 disassembly in rad-51::FLAG mutants lead to formation of chromosomal fragments, similar in their magnitude to ones observed in rad-51 or rad-54.L null mutants. However, rad-51::FLAG mutants (unlike a rad-51 null, GFP::rad-51 or rad-54.L null mutants) displayed no defects in the formation of crossover-designated sites (via GFP::COSA-1 localization). Given that rad-51::FLAG worms show checkpoint activation and chromosomal fragments, these results suggest that crossover repair concludes normally, while the noncrossover pathway is perturbed. This is strikingly different from rad-51::degron and GFP::rad-51 strains, which are proficient or deficient in both pathways, respectively. These results suggest that noncrossovers vs crossovers have distinct recombination intermediates and diverge prior to RAD-51 disassembly.

• Klíčová slova
• C. elegans, RAD51, crossover, meiosis,
• MeSH
• alely MeSH
• crossing over (genetika) MeSH
• dvouřetězcové zlomy DNA MeSH
• homologní rekombinace MeSH
• křížová struktura DNA * genetika   metabolismus   MeSH
• meióza MeSH
• oprava DNA MeSH
• proteiny Caenorhabditis elegans MeSH
• rekombinasa Rad51 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• křížová struktura DNA * MeSH
• proteiny Caenorhabditis elegans MeSH
• rad-51 protein, C elegans MeSH Prohlížeč
• rekombinasa Rad51 * MeSH

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

• MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• jednovláknová DNA * metabolismus   genetika   MeSH
• lidé MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• rekombinasa Rad51 * metabolismus   genetika   MeSH
• replikace DNA * genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• jednovláknová DNA * MeSH
• multiproteinové komplexy MeSH
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 * 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
• 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

OBJECTIVES: DNA repair proteins have emerged as potential predictors for immunotherapy response alongside PD-L1 expression, tumor-infiltrating lymphocytes (TILs) and tumor mutational burden. We analyzed expression of PD-L1, TILs count and expression of the homologous recombination (HR) protein RAD51, as potential prognostic factors in patients with resected non-small-cell lung carcinoma (NSCLC). MATERIALS AND METHODS: Discovery set included 96 NSCLC patients from the University Hospital Olomouc (Czech Republic) and a replication set included 1109 NSCLC patients from University Hospital Zurich (Switzerland). Tissue microarrays (TMAs) were stained using the automated staining platform Ventana Benchmark Ultra with antibodies against RAD51,CD3, CD8, CD68 and PD-L1. RESULTS: Loss of nuclear RAD51 protein was associated with high TILs (r=-0.25, p = 0.01) and PD-L1 status (10.6 vs. 2.4 %, p = 0.012) in patients receiving neoadjuvant chemo-/radiotherapy (CT/RT). In silico analysis from the TCGA data set showed a negative relationship between RAD51 mRNA expression and CD45 (r = ‒0.422, p < 0.0001), CD68 (r = ‒0.326, p < 0.001), CD3 (r = ‒0.266, p < 0.001) and CD8 (r = ‒0.102, p < 0.001). RAD51 low/PD-L1 high patients were clustered as separate entity in the replication set and in TCGA dataset. High TILs status was significantly associated with improved OS in the replication set (unadjusted HR = 0.57, 95 % CI 0.42-0.76, p < 0.001). Similar results have been seen for CD3, CD8 and CD68. CONCLUSIONS: In conclusion, RAD51 nuclear loss is weakly associated with increased TILs and high PD-L1 at the time of surgery in curatively resected NSCLC and after prior exposure to neoadjuvant chemo- or radiotherapy. Both high TILs and RAD51 nuclear loss were confirmed as independent prognostic factors in curatively resected NSCLC.

• Klíčová slova
• Neoadjuvant, PD-L1, Prognostic, RAD51, Tumor infiltrating lymphocytes,
• MeSH
• antigeny CD274 genetika   MeSH
• lidé MeSH
• nádory plic * genetika   terapie   MeSH
• nemalobuněčný karcinom plic * genetika   terapie   MeSH
• oprava DNA MeSH
• prognóza MeSH
• rekombinasa Rad51 genetika   MeSH
• tumor infiltrující lymfocyty MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Švýcarsko MeSH
• Názvy látek
• antigeny CD274 MeSH
• CD274 protein, human MeSH Prohlížeč
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 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.

• Klíčová slova
• DNA ligase IV, MUS81, R-loop, RECQ5, replication fork reversal, replication restart, replication stress, transcription-replication conflict,
• 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
• Názvy látek
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA vazebné proteiny MeSH
• DNA-ligasy MeSH
• DNA-polymerasa III MeSH
• endodeoxyribonukleasy MeSH
• endonukleasy MeSH
• helikasy RecQ MeSH
• Lig4 protein, Arabidopsis MeSH Prohlížeč
• MUS81 protein, human MeSH Prohlížeč
• RAD51 protein, human MeSH Prohlížeč
• RAD52 protein, human MeSH Prohlížeč
• RECQL protein, human MeSH Prohlížeč
• RECQL5 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• XRCC4 protein, human MeSH Prohlížeč

Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.

• Klíčová slova
• Physcomitrella patens, RAD51, RTEL1, Sog One-Like, genome stability, ribosomal RNA genes, telomere,
• MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• genetické lokusy MeSH
• mechy enzymologie   genetika   MeSH
• mutace MeSH
• nestabilita genomu * MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• RNA ribozomální 5S genetika   MeSH
• RNA ribozomální genetika   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• telomery genetika   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• rekombinasa Rad51 MeSH
• ribozomální DNA MeSH
• RNA ribozomální 18S MeSH
• RNA ribozomální 5S MeSH
• RNA ribozomální MeSH
• RNA, ribosomal, 45S MeSH Prohlížeč
• rostlinné proteiny MeSH
• transkripční faktory 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

Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.

• Klíčová slova
• Fanconi anemia, RAD51, recombination, replication,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• Fanconiho anemie genetika   MeSH
• homologní protein MRE11 metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikace DNA * MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• Xenopus MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• homologní protein MRE11 MeSH
• MRE11 protein, human MeSH Prohlížeč
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH

Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors, and increased DNA repair capacity is a hallmark mechanism of resistance not only to DNA-damaging treatments such as cytotoxic drugs and radiotherapy, but also to small molecule targeted drugs such as inhibitors of poly-ADP ribose polymerase (PARP). Hence, there is substantial medical need for potent and convenient biomarkers of individual response to DNA-targeted treatment in personalized cancer care. RAD51 is a highly conserved protein that catalyzes DNA repair via homologous recombination, a major DNA repair pathway which directly modulates cellular sensitivity to DNA-damaging treatments. The clinical and biological significance of RAD51 protein expression is still under investigation. Pre-clinical studies consistently show the important role of nuclear RAD51 immunoreactivity in chemo- and radioresistance. Validating data from clinical trials however is limited at present, and some clinical studies show controversial results. This review gives a comprehensive overview on the current knowledge about the prognostic and predictive value of RAD51 protein expression and genetic variability in patients with solid malignancies.

• Klíčová slova
• RAD51, prognosis, radiotherapy, “BRCAness,” chemotherapy,
• MeSH
• chemorezistence genetika   MeSH
• DNA nádorová účinky léků   účinky záření   MeSH
• geny BRCA1 MeSH
• geny BRCA2 MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádory farmakoterapie   genetika   metabolismus   radioterapie   MeSH
• oprava DNA genetika   MeSH
• PARP inhibitory terapeutické užití   MeSH
• polymorfismus genetický MeSH
• proliferace buněk genetika   MeSH
• rekombinační oprava DNA genetika   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• tolerance záření genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA nádorová MeSH
• nádorové biomarkery MeSH
• PARP inhibitory MeSH
• rekombinasa Rad51 MeSH