Flap endonuclease 1 (FEN1)-dependent long-patch repair has been considered a minor sub-pathway of DNA single-strand break repair (SSBR), activated only when short-patch repair is not feasible. However, the significance of long-patch repair in living cells remains unclear. Here, we employed human RPE-1 cells with FEN1 deletion to compare the requirements for short- and long-patch pathways for the rapid repair of various types of DNA single-strand breaks (SSBs). We found that SSBs arising from abortive topoisomerase 1 activity are repaired efficiently without FEN1. In contrast, the rapid repair of SSBs arising during base excision repair following treatment with methyl methanesulphonate (MMS) or following treatment with hydrogen peroxide (H2O2) exhibits an unexpectedly high dependence on FEN1. Indeed, in G1 phase, FEN1 deletion slows the rate of SSBR to a similar or even greater extent than deletion of the short-patch repair proteins XRCC1 or POLβ. As expected, the combined deletion of FEN1 with XRCC1 or POLβ has an additive or synergistic effect, severely attenuating SSBR rates after MMS or H2O2 exposure. These data highlight an unanticipated requirement for FEN1 in the rapid repair of SSBs in human cells, challenging the prevailing view that long-patch repair is a minor sub-pathway of SSBR.

• MeSH
• "flap" endonukleasy * genetika   fyziologie   metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny genetika   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• G1 fáze * genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• methylmethansulfonát toxicita   MeSH
• oprava DNA * MeSH
• peroxid vodíku farmakologie   toxicita   MeSH
• protein XRCC1 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy * MeSH
• DNA vazebné proteiny MeSH
• DNA-topoisomerasy I MeSH
• FEN1 protein, human MeSH Prohlížeč
• methylmethansulfonát MeSH
• peroxid vodíku MeSH
• protein XRCC1 MeSH
• XRCC1 protein, human MeSH Prohlížeč

Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.

• MeSH
• Cas proteiny genetika   metabolismus   MeSH
• CRISPR-Cas systémy * genetika   MeSH
• editace genu * metody   MeSH
• endodeoxyribonukleasy genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• integrasy * genetika   metabolismus   MeSH
• lidé MeSH
• sekvence CRISPR * MeSH
• vodící RNA, systémy CRISPR-Cas genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• Cas proteiny MeSH
• Cas12a protein MeSH Prohlížeč
• Cre recombinase MeSH Prohlížeč
• endodeoxyribonukleasy MeSH
• integrasy * MeSH
• vodící RNA, systémy CRISPR-Cas MeSH

Helicases and endonucleases play crucial roles in genome maintenance by unwinding or cleaving various forms of DNA and RNA structures in order to facilitate essential biological processes, such as DNA replication and recombination. Here, we identified fission yeast Dbl2 as a potential interactor of several complexes that exhibit either helicase or endonuclease activity, namely Fml1-MHF, SCFFbh1, Rqh1-Top3-Rmi1, and Mus81-Eme1. In vitro, Dbl2 binds to DNA, with a preference for branched molecules, such as D-loops, mobile Holliday junctions, and fork structures, making it a good candidate to play a central role in modulating the activity of helicases and endonucleases during replication and recombination repair. Previously, we showed that Dbl2 recruits Fbh1 to the ongoing homologous recombination sites, affecting the Rad51-nucleofilament. In this study, we determined that deleting dbl2 in an fbh1Δ background did not increase sensitivity to DNA-damaging agents or the frequency of Tf2 ectopic recombination. Therefore, Dbl2 and Fbh1 might be involved in the same molecular pathway, maintaining genome integrity by hindering ectopic recombination at repetitive elements.

• Klíčová slova
• Schizosaccharomyces pombe, DNA repair, Dbl2, Helicases, Homologous recombination,
• MeSH
• DNA fungální metabolismus   genetika   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-helikasy * metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• poškození DNA MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• replikace DNA MeSH
• Schizosaccharomyces pombe - proteiny * metabolismus   genetika   MeSH
• Schizosaccharomyces * genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA fungální MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy * MeSH
• endonukleasy * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH

The polymerase acidic (PA) subunit of the influenza virus, an endonuclease of the RNA-dependent RNA polymerase, represents a viable target for anti-influenza therapies, as evidenced by the efficacy of the FDA-approved drug Xofluza. A characteristic feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions within the enzyme's catalytic site. Previously, our studies identified luteolin and its C-8-glucoside orientin as potent endonuclease inhibitors. This report details our subsequent investigation into the structural modifications of the phenyl moiety attached to the C-8 position of luteolin. The inhibitory potencies (IC50 values) quantified with AlphaScreen technology indicated that substituting the C-8 glucose moiety of orientin resulted in compounds with comparable inhibitory potency. From a series of eighteen compounds, acid 12 with 3-carboxylphenyl moiety at the C-8 position was the most potent inhibitor with nanomolar potency.

• Klíčová slova
• Endonuclease, Flavonoid, Influenza, Inhibitors, RNA polymerase,
• MeSH
• antivirové látky * farmakologie   chemická syntéza   chemie   MeSH
• endonukleasy * antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů * farmakologie   chemická syntéza   chemie   MeSH
• luteolin * farmakologie   chemická syntéza   chemie   MeSH
• molekulární struktura MeSH
• racionální návrh léčiv * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• endonukleasy * MeSH
• inhibitory enzymů * MeSH
• luteolin * MeSH

BACKGROUND: The unfolded protein response (UPR) enables myeloma cells to overcome the stress conditions arising from excessive proteosynthesis and thus provides a survival advantage for myeloma cells. Extramedullary disease is a more aggressive form of multiple myeloma in which myeloma cells lose their dependence on the bone marrow microenvironment and are able to infiltrate other tissues and organs. The pathogenesis of extramedullary disease is not fully elucidated yet. The aim of this study was to determine whether there is a difference in the expression of UPR-related genes between bone marrow plasma cells from multiple myeloma and extramedullary disease patients. MATERIALS AND METHODS: Gene expression of six genes involved in UPR (ERN1, DDIT3, EIF2AK3, TUSC3, XBP1, HSPA5) was analyzed by quantitative reverse transcription polymerase chain reaction. In total, 76 bone marrow plasma cell samples were used, of which 44 were from patients with multiple myeloma and 32 from patients with extramedullary disease. RESULTS: A statistically significant difference was observed between the multiple myeloma and extramedullary disease groups regarding the expression of HSPA5, DDIT3, EIF2AK3, and ERN1 genes. However, in the case of XBP1 and TUSC3 genes, no statistically significant difference in the expression was found. Several statistically significant correlations between the expression levels of the analyzed genes and the clinical data of the patients were observed as well. CONCLUSION: Our results suggest the importance of UPR in the pathogenesis of extramedullary disease. UPR appears to be a promising avenue for further research.

• Klíčová slova
• Kahler-Pick law, Multiple myeloma, Plasma cells, extramedullary disease, unfolded protein response,
• MeSH
• chaperon endoplazmatického retikula BiP MeSH
• endoribonukleasy genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mnohočetný myelom * genetika   metabolismus   patologie   MeSH
• plazmatické buňky metabolismus   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteiny teplotního šoku genetika   MeSH
• senioři MeSH
• signální dráha UPR * genetika   MeSH
• transkripční faktor CHOP genetika   MeSH
• transkripční faktory RFX MeSH
• XBP1 genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chaperon endoplazmatického retikula BiP MeSH
• DDIT3 protein, human MeSH Prohlížeč
• EIF2AK3 protein, human MeSH Prohlížeč
• endoribonukleasy MeSH
• ERN1 protein, human MeSH Prohlížeč
• HSPA5 protein, human MeSH Prohlížeč
• kinasa eIF-2 MeSH
• protein-serin-threoninkinasy MeSH
• proteiny teplotního šoku MeSH
• transkripční faktor CHOP MeSH
• transkripční faktory RFX MeSH
• XBP1 protein, human MeSH Prohlížeč
• XBP1 MeSH

Here we investigated cytotoxicity and DNA and protein binding of an iodido analog of picoplatin, the cis-ammine-diiodido(2-methylpyridine)platinum(II) complex (I-picoplatin). I-picoplatin (IC50 = 3.7-12.4 μM) outperforms picoplatin (IC50 = 11.8-22.6 μM) in the human cancer cell lines used and shows a greater ability to overcome the cisplatin resistance of A2780 ovarian cancer cells than does picoplatin. I-picoplatin also induces different cell cycle changes (reduced S-phase fraction and an increase in the G2/M phase arrest) in HeLa cervical carcinoma cells compared to both picoplatin and cisplatin. Binding of the metal compound to DNA model systems was investigated by ethidium bromide displacement assay and circular dichroism. Its reactivity with lysozyme (HEWL) and pancreatic RNase A was studied by X-ray diffraction and mass spectrometry experiments. I-picoplatin binds the DNA double helix and is able to retain the 2-methylpyridine ligand and at least one of the two iodido ligands when bound to the two proteins. Various Pt-containing moieties, including one based on the isomerized structure of I-picoplatin, coordinate the His and Met residues. A low-resolution structure of the I-picoplatin/human serum albumin (HSA) adduct has also been solved. The side chains of His146, Met289, and Met329 are the primary binding sites of the I-picoplatin moieties on HSA.

• MeSH
• DNA * metabolismus   chemie   MeSH
• lidé MeSH
• lidský sérový albumin * metabolismus   chemie   MeSH
• molekulární struktura MeSH
• muramidasa * metabolismus   chemie   MeSH
• nádorové buněčné linie MeSH
• organoplatinové sloučeniny * chemie   farmakologie   chemická syntéza   metabolismus   MeSH
• pankreatická ribonukleasa * metabolismus   chemie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky * farmakologie   chemie   chemická syntéza   metabolismus   MeSH
• screeningové testy protinádorových léčiv MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA * MeSH
• lidský sérový albumin * MeSH
• muramidasa * MeSH
• organoplatinové sloučeniny * MeSH
• pankreatická ribonukleasa * MeSH
• protinádorové látky * MeSH

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 influenza RNA-dependent RNA polymerase harbours an endonuclease subunit characterized by a catalytic site housing two divalent metal ions. By effectively chelating both Mg2+ and Mn2+ ions, a small-molecule inhibitor with a metal-binding pharmacophore can halt endonuclease activity. Herein, two 3'-dehydroxypurpurogallin-4-carboxamide series, namely twelve C-4' unsubstituted and twelve C-4' phenyl substituted congeners were designed and prepared to be tested as inhibitors of the metal-dependent viral enzyme. These inhibitors were accessed through the chemoenzymatic reaction of gallic acid with either pyrocatechol or phenylpyrocatechol moderated by laccase, followed by amidation. Experimental IC50 values were determined using AlphaScreen technology, with the most potent inhibitors exhibiting IC50 values around 0.35 μM. Using X-ray crystallography, we analyzed structure of the endonuclease in complex with one potent 3'-dehydroxypurpurogallin-carboxamide at 2.0 Å resolution, revealing the coordination of the compound's triad of oxygen atoms with the two metal ions in the influenza A endonuclease active site.

• Klíčová slova
• Chemical synthesis, Endonuclease, Enzyme inhibition, Influenza, Metalloenzymes,
• MeSH
• amidy * chemie   farmakologie   chemická syntéza   MeSH
• antivirové látky * chemická syntéza   farmakologie   chemie   MeSH
• endonukleasy * antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů * chemická syntéza   chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• kyselina gallová * chemie   farmakologie   analogy a deriváty   chemická syntéza   MeSH
• lidé MeSH
• molekulární struktura MeSH
• virus chřipky A * enzymologie   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
• amidy * MeSH
• antivirové látky * MeSH
• endonukleasy * MeSH
• inhibitory enzymů * MeSH
• kyselina gallová * MeSH