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Background Ubiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development. Results We developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones. Conclusions These approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine–guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein’s affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation 1 , 2 , 3 – 4 . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor 3 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses, we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the single-stranded DNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1. A head-to-head double-ring complex of the human multifunctional DNA repair protein RAD52 mediates protection of stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor.

A DNS a sejtciklus során folyamatosan károsító tényezőknek van kitéve. Ezek között a tényezők között találunk exogén és endogén ágenseket, mint például reaktív oxigén gyökök, UV sugárzás, nitrogén-mustár, formaldehid, stb., amelyek különféle módon rongálják a DNS-t. A DNS-károsodások a sejtekben olyan különböző mechanizmusokat aktiválnak, melyeknek az a szerepe, hogy kijavítsák a keletkezett hibákat, ezért ezeket együttesen DNS-hibajavító mechanizmusoknak nevezzük. Jelen dolgozatban elsősorban a posztreplikációs hibajavító mechanizmussal foglalkozunk, amelyik az elakadt replikációs villánál indul. Ennek első fázisában a PCNA poszttranszlációs módosításon esik át, ami lehet ubikvitinálás vagy SUMOiláció, melyek révén egyrészt egyéb fehérjéket aktivál, másrészt meghatározza, hogy mely útvonalon haladjon a DNS-hibajavítás mechanizmusa. A PCNA molekula az elakadt replikációs villánál az UBA1 ubikvitinaktiváló enzim, a Rad6 ubikvitin-konjugáz és a Rad18 ubikvitin-ligáz hatására a K164-es lizinjén monoubikvitinálódik. A monoubikvitinált PCNA a polimerázok egy speciális családját, az ún. TLS (transzléziós szintézis) polimerázokat toborozza a hibához. Ezek a polimerázok hibásan írják át a DNS-szálat, ami mutagenezishez és végső soron karcinogenezishez vezet. Azt a folyamatot, amely hibás DNS-átírást eredményez és a TLS polimerázok révén valósul meg, transzléziós DNS szintézisnek nevezzük.Munkám során egy nagy áteresztőképességű szűrő módszert fejlesztettem ki, amely képes azonosítani a PCNA ubikvitinálást befolyásoló molekulákat. Ezt követően tovább fejlesztettük a felállított rendszert a jóval érzékenyebb Alpha technológiához. Célunk a reakciókban használt komponensek koncentrációjának csökkentése és az azt követő kimutatási lépés optimalizálása volt, az Uba1-ubikvitin tioészter, a Rad6-ubikvitin tioészter, a Rad6-Rad18 interakciós, valamint a Rad18 autoubikvitilálási assay-k esetén.A tesztek során sikeresen azonosítottunk egy a zöld teában lévő vegyületet, az EGCG-t (epigallocatechin gallát), amely az Uba1-ubikvitin tioészter komplex kialakulásának potenciális inhibitora, és ezáltal a PCNA ubikvitinálás hatékony gátlója lehet. Ezt követően az EGCG molekulát in vitroteszteltük HEK293 sejtvonalban, ahol sejtosztódásgátló hatást mutatott. A sejtmagi mágneses rezonancia (NMR) tesztek igazolták az EGCG és az Uba1 közvetlen kapcsolódását. Az EGCG és az Uba1 közötti kölcsönhatás reverzibilis, amit centrifugális-kimosási kísérletekkel vizsgáltunk. Az Uba1 fehérje HEK293 sejtekben történő túltermeltetése védelmet nyújtott az EGCG gátló hatásával szemben. Továbbá kimutattuk, hogy az EGCG globálisan gátolja az ubikvitinálást a sejtekben.Vizsgáltuk az EGCG és rokon molekuláinak hatását a PCNA ubikvitinálásra, az Uba1- ubikvitin tioészter komplexre és a HEK293 sejtek túlélésére. A kísérletekhez használt catechin galloyl észter EGCG és ECG (epigallocatechin) a tesztek során végig aktívnak mutatkozott. A nem-észterezett cathechin és (−)-ECG csupán alacsony aktivitást mutatott úgy a PCNA ubikvitinálás, mint az Uba1-ubikvitin tioészter formálás alkalmával, míg az (−)-epicatechin (EC) és a (+)-catechin (Cat) mindkét esetben inaktív maradt. Az EC-vel és a Cat-tal ellentétben az ECG-nek mérhető citosztatikus/citotoxikus hatása is volt. Az ECG negatív hatását a sejtek túlélésére nem befolyásolta az Uba1 vagy az ubikvitin túltermeltetése. A szabad galluszsav (GA) és az n-propyl gallát (PG), egy rövid, egyenes láncú alkil-galloil-észter, szintén inaktív volt a PCNA ubikvitinálás és az Uba1-ubikvitin tioészter komplex formációs tesztek során, de a sejtek túlélését kis mértékben befolyásolták. Ezt a gátló hatást az Uba1 vagy az ubiquitin túlzott expressziója nem mérsékelte. A hosszabb egyenes láncú alkil galloil észterek, az n-octil gallát (OG) és az n-dodecil (lauril) gallát (DG), minden vizsgált tesztben aktívak voltak, és az Uba1 vagy az ubikvitin túltermelése védelmet nyújtott a sejteknek a túlélésre gyakorolt negatív hatástól.

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure–activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

The human genome contains numerous repetitive nucleotide sequences that display a propensity to fold into non-canonical DNA structures including G-quadruplexes (G4s). G4s have both positive and negative impacts on various aspects of nucleic acid metabolism including DNA replication, DNA repair and RNA transcription. Poly (ADP-ribose) polymerase (PARP1), an important anticancer drug target, has been recently shown to bind a subset of G4s, and to undergo auto-PARylation. The mechanism of this interaction, however, is poorly understood. Utilizing Mass Photometry (MP) and single-molecule total internal reflection fluorescence microscopy (smTIRFM), we demonstrate that PARP1 dynamically interacts with G4s with a 1:1 stoichiometry. Interaction of a single PARP1 molecule with nicked DNA or DNA containing G4 and a primer-template junction is sufficient to activate robust auto-PARylation resulting in the addition of poly (ADP-ribose) chains with molecular weight of several hundred kDa. Pharmacological PARP inhibitors EB-47, Olaparib and Veliparib differently affect PARP1 retention on G4-containing DNA compared to nicked DNA.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al . synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro , and to display better selectivity towards G4s than the previously published BG4 antibody. To get insight into the G4P-G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al . synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro , and to display better selectivity towards G4s than the previously published BG4 antibody. To get insight into the G4P-G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1. Using cryo-EM, biochemical and single-molecule approaches we show that the structure of stalled DNA replication fork promotes a unique two-ring organization of human RAD52 protein which remodels the fork via DNA strand exchange.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Polycrystalline Y-type hexagonal ferrite sample with composition Ba 2− x Pb x Co 1.5 Zn 0.5 Fe 12 O 22 (0 <  x  < 0.4) have been prepared via sol–gel auto-combustion method with citric acid as a chelating agent. The synthesized samples were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), vibrating sample magnetometry, and P – E loop tracer. Single-phase of Y-type hexaferrite has been confirmed by XRD. The crystalline nature with hexagonal platelet-like morphology was confirmed by structural and morphological analysis. The dielectric constant ( ε ′) increases with Pb concentration and maximum dispersion is obtained for x  = 0.3 sample. The power law dependence of ac conductivity with exponent ( n ) value about 0.229 to 0.285, suggests the conduction mechanism via polaron hooping. P – E loop analysis demonstrated the increase in remnant polarization, saturation polarization value with increase of Pb (up to x  = 0.2) and shows a decreasing trend for higher Pb content. M – H curves reveal the soft magnetic behavior of the prepared samples. The coercivity ( H c ) increases with an increase of Pb content and x  = 0.3 sample exhibits the maximum value of coercivity of about ~ 107 Oe. The room temperature magnetoelectric coupling is evidenced with a second-order coupling coefficient ( β ) of 4.3 × 10 −6  μV/cm-Oe 2 for x  = 0.3 sample, which endorses this material as a potential candidate for multistate memory devices.