Chiroptical studies of the SpyCas9 protein are extremely rare. Nondestructive methods are needed to characterize its active ribonucleoprotein form. Using Raman optical activity (ROA) and circularly polarized luminescence (CPL), we present a new approach to detect key biomolecules involved in CRISPR-Cas technology while preserving their original nucleolytic activity.

• MeSH
• CRISPR-Cas systémy MeSH
• luminiscence MeSH
• protein Cas9 * metabolismus   chemie   MeSH
• Ramanova spektroskopie MeSH
• roztoky MeSH
• stereoizomerie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein Cas9 * MeSH
• roztoky MeSH

Chemoresistance poses one of the most significant challenges of cancer therapy. Carboplatin (CbPt) is one of the most used chemotherapeutics in ovarian cancer (OVC) treatment. MRE11 constitutes a part of homologous recombination (HR), which is responsible for the repair of CbPt-induced DNA damage, particularly DNA crosslinks. The study's main aim was to address the role of HR in CbPt chemoresistance in OVC and to evaluate the possibility of overcoming CbPt chemoresistance by Mirin-mediated MRE11 inhibition in an OVC cell line. Lower expression of MRE11 was associated with better overall survival in a cohort of OVC patients treated with platinum drugs (TCGA dataset, P < 0.05). Using in vitro analyses, we showed that the high expression of HR genes drives the CbPt chemoresistance in our CbPt-resistant cell line model. Moreover, the HR inhibition by Mirin not only increased sensitivity to carboplatin (P < 0.05) but also rescued the sensitivity in the CbPt-resistant model (P < 0.05). Our results suggest that MRE11 inhibition with Mirin may represent a promising way to overcome OVC resistance. More therapy options will ultimately lead to better personalized cancer therapy and improvement of patients' survival.

• Klíčová slova
• DNA repair, MRE11, cancer therapy, chemoresistance, ovarian cancer,
• MeSH
• chemorezistence * genetika   účinky léků   MeSH
• homologní protein MRE11 * genetika   metabolismus   antagonisté a inhibitory   MeSH
• karboplatina * farmakologie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory vaječníků * farmakoterapie   genetika   patologie   MeSH
• protinádorové látky * farmakologie   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• rekombinační oprava DNA * účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• homologní protein MRE11 * MeSH
• karboplatina * MeSH
• MRE11 protein, human MeSH Prohlížeč
• protinádorové látky * MeSH

MRE11 nuclease is a central player in signaling and processing DNA damage, and in resolving stalled replication forks. Here, we describe the identification and characterization of new MRE11 inhibitors MU147 and MU1409. Both compounds inhibit MRE11 nuclease more specifically and effectively than the relatively weak state-of-the-art inhibitor mirin. They also abrogate double-strand break repair mechanisms that rely on MRE11 nuclease activity, without impairing ATM activation. Inhibition of MRE11 also impairs nascent strand degradation of stalled replication forks and selectively affects BRCA2-deficient cells. Herein, we illustrate that our newly discovered compounds MU147 and MU1409 can be used as chemical probes to further explore the biological role of MRE11 and support the potential clinical relevance of pharmacological inhibition of this nuclease.

• Klíčová slova
• BRCA2, FEN1, MRE11 inhibitor, nuclease,
• MeSH
• homologní protein MRE11 * metabolismus   antagonisté a inhibitory   MeSH
• inhibitory enzymů * farmakologie   chemie   chemická syntéza   MeSH
• lidé MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• oprava DNA účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• homologní protein MRE11 * MeSH
• inhibitory enzymů * MeSH
• MRE11 protein, human MeSH Prohlížeč

The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

• Klíčová slova
• MRE11, NBN, RAD50, germline variants, meta‐analysis,
• MeSH
• DNA vazebné proteiny genetika   MeSH
• enzymy opravy DNA genetika   MeSH
• genetická predispozice k nemoci * MeSH
• homologní protein MRE11 * genetika   MeSH
• hydrolasy působící na anhydridy kyselin * genetika   MeSH
• jaderné proteiny * genetika   MeSH
• lidé MeSH
• nádory * genetika   MeSH
• proteiny buněčného cyklu * genetika   MeSH
• zárodečné mutace * MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• systematický přehled MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• enzymy opravy DNA MeSH
• homologní protein MRE11 * MeSH
• hydrolasy působící na anhydridy kyselin * MeSH
• jaderné proteiny * MeSH
• MRE11 protein, human MeSH Prohlížeč
• NBN protein, human MeSH Prohlížeč
• proteiny buněčného cyklu * MeSH
• RAD50 protein, human MeSH Prohlížeč

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• "flap" endonukleasy metabolismus   genetika   MeSH
• DEAD-box RNA-helikasy * metabolismus   genetika   MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• DNA-helikasy * metabolismus   genetika   MeSH
• DNA-ligasa ATP metabolismus   genetika   MeSH
• DNA metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• lidé MeSH
• multifunkční enzymy * metabolismus   genetika   MeSH
• R-smyčka * MeSH
• replikace DNA * MeSH
• RNA-helikasy * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DEAD-box RNA-helikasy * MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy * MeSH
• DNA-ligasa ATP MeSH
• DNA MeSH
• endonukleasy * MeSH
• multifunkční enzymy * MeSH
• MUS81 protein, human MeSH Prohlížeč
• RNA-helikasy * MeSH
• SETX protein, human MeSH Prohlížeč

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

Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.

• MeSH
• chromatin * metabolismus   MeSH
• DNA vazebné proteiny metabolismus   genetika   MeSH
• dvouřetězcové zlomy DNA * MeSH
• endodeoxyribonukleasy * metabolismus   genetika   MeSH
• exodeoxyribonukleasy metabolismus   genetika   MeSH
• meióza * genetika   MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• DNA vazebné proteiny MeSH
• endodeoxyribonukleasy * MeSH
• exodeoxyribonukleasy MeSH
• meiotic recombination protein SPO11 MeSH Prohlížeč
• MRE11 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny * MeSH
• Spo11 protein, S cerevisiae MeSH Prohlížeč

The guidelines for clinical practice for carriers of pathogenic variants in clinically relevant genes predisposing to Lynch syndrome and colorectal cancer define the steps of primary and secondary prevention that should be provided to the individuals at high risk of developing hereditary cancer in the Czech Republic. The drafting of the guidelines was organized by the Oncogenetics Working Group of the Society for Medical Genetics and Genomics of J. E. Purkyně Czech Medical Society, in cooperation with representatives of oncology, oncogynecology, and gastroenterology. The guidelines are based on the current recommendations of the National Comprehensive Cancer Network (NCCN), European Society of Medical Oncology (ESMO) and take into account the capacity of the Czech healthcare system.

• Klíčová slova
• MLH1, MSH2/EPCAM, MSH6, PMS2, consensus, germline mutation carriers, guidelines for clinical practice,
• MeSH
• adhezní molekula epiteliálních buněk * genetika   MeSH
• dědičné nepolypózní kolorektální nádory * genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• genetická predispozice k nemoci * MeSH
• homolog 2 proteinu MutS * genetika   MeSH
• lidé MeSH
• mismatch repair endonukleáza PMS2 genetika   MeSH
• MutL homolog 1 * genetika   MeSH
• zárodečné mutace * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• směrnice pro lékařskou praxi MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• adhezní molekula epiteliálních buněk * MeSH
• DNA vazebné proteiny MeSH
• EPCAM protein, human MeSH Prohlížeč
• G-T mismatch-binding protein MeSH Prohlížeč
• homolog 2 proteinu MutS * MeSH
• mismatch repair endonukleáza PMS2 MeSH
• MLH1 protein, human MeSH Prohlížeč
• MSH2 protein, human MeSH Prohlížeč
• MutL homolog 1 * MeSH
• PMS2 protein, human MeSH Prohlížeč

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

• Klíčová slova
• BRCA2, DNA Repair, EXO1, FEN1, PARG,
• MeSH
• "flap" endonukleasy genetika   metabolismus   terapeutické užití   MeSH
• enzymy opravy DNA genetika   MeSH
• exodeoxyribonukleasy genetika   MeSH
• glykosidhydrolasy genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * genetika   metabolismus   MeSH
• nádory * farmakoterapie   genetika   MeSH
• oprava DNA MeSH
• PARP inhibitory farmakologie   MeSH
• poškození DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• enzymy opravy DNA MeSH
• EXO1 protein, human MeSH Prohlížeč
• exodeoxyribonukleasy MeSH
• FEN1 protein, human MeSH Prohlížeč
• glykosidhydrolasy MeSH
• nádorový supresorový protein p53 * MeSH
• PARP inhibitory MeSH

Upper tract urothelial carcinoma (UTUC) is the third most common malignancy associated with Lynch syndrome (LS). The current European urology guidelines recommend screening for LS in patients with UTUC up to the age of 60 years. In this study, we examined a cohort of patients with UTUC for potential association with LS in order to establish the sensitivity of current guidelines in detecting LS. A total of 180 patients with confirmed diagnosis of UTUC were enrolled in the study during a 12-year period (2010-2022). Loss of DNA-mismatch repair proteins (MMRp) expression was identified in 15/180 patients (8.3%). Germline analysis was eventually performed in 8 patients confirming LS in 5 patients (2.8%), including 4 germline mutations in MSH6 and 1 germline mutation in MSH2. LS-related UTUC included 3 females and 2 males, with a mean age of 66.2 years (median 71 years, range 46-75 years). Four of five LS patients (all with MSH6 mutation) were older than 65 years (mean age 71.3, median 72 years). Our findings indicate that LS-associated UTUCs can occur in patients with LS older than 60 years. In contrast to previous studies which used mainly highly pre-selected populations with already diagnosed LS, the most frequent mutation in our cohort involved MSH6 gene. All MSH6 mutation carriers were > 65 years, and UTUC was the first LS manifestation in 2/4 patients. Using current screening guidelines, a significant proportion of patients with LS-associated UTUC may be missed. We suggest universal immunohistochemical MMRp screening for all UTUCs, regardless of age and clinical history.

• Klíčová slova
• Immunohistochemistry, Lynch syndrome, MMR, Screening algorithm, Upper urinary tract, Urothelial carcinoma,
• MeSH
• dědičné nepolypózní kolorektální nádory * diagnóza   genetika   patologie   MeSH
• karcinom z přechodných buněk * genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mismatch repair endonukleáza PMS2 genetika   MeSH
• MutL homolog 1 genetika   MeSH
• nádory močového měchýře * MeSH
• oprava chybného párování bází DNA MeSH
• senioři MeSH
• urologie * MeSH
• zárodečné mutace MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mismatch repair endonukleáza PMS2 MeSH
• MutL homolog 1 MeSH