DNS helikáz motorok enzimológiája = Motor enzymology of DNA helicases

A projekt során a genom épségének fenntartásában központi szerepet játszó RecQ-családbeli DNS-helikázok közül az E. coli RecQ enzim és a humán Bloom-szindróma helikáz (BLM) működési mechanizmusát derítettük fel élvonalbeli gyorskinetikai, spektroszkópiai és egyedimolekula-biofizikai technikákkal. Kidolgoztunk egy analitikai eljárást, amelynek segítségével a nukleinsavak mentén haladó motorfehérjék legfontosabb működési jellemzői tranziens kinetikai kísérletsorozatokban meghatározhatók. Meghatároztuk és publikáltuk a RecQ és BLM helikázok egyszálú DNS-en történő transzlokációjának mechanizmusát. Meghatároztuk a BLM helikáz legfontosabb szerkezeti elemeinek szerepét a DNS-átalakító aktivitásokban, valamint leírtuk az enzim szubsztrát-indukált oligomerizációs mechanizmusát. A DNS-szálszétválasztás aktív duplex-destabilizációval megvalósuló mechanizmusát egyedimolekula-vizsgálatokkal derítettük fel. Leírtuk a RecQ helikázok más rekombinációs fehérjékkel történő együttműködésének számos új aspektusát. | In this project we elucidated the mechanism of action of two RecQ-family DNA helicases (E. coli RecQ and human Bloom’s syndrome (BLM) helicases), which are essential players in maintaining the integrity of the genome. In this work we applied cutting-edge rapid transient kinetic and single molecule biophysical techniques. We developed an analytical method suitable for the determination of all key functional parameters of DNA-based motor enzymes. We elucidated the mechanism of translocation of RecQ and BLM helicases along single-stranded DNA. We identified the roles of the most important structural elements of BLM in the DNA-restructuring activities, and described the substrate-induced oligomerization mechanism of the enzyme. By using single molecule techniques we elucidated the physical mechanism of DNA strand separation that involves active destabilization of DNA duplexes. We also revealed several novel aspects of the cooperation of RecQ halicases with other proteins during homologous recombination processes

Processive translocation mechanism of the human Bloom’s syndrome helicase along single-stranded DNA

BLM, one of the human RecQ helicases, plays a fundamental role in homologous recombination-based error-free DNA repair pathways, which require its translocation and DNA unwinding activities. Although translocation is essential in vivo during DNA repair processes and it provides a framework for more complex activities of helicases, including strand separation and nucleoprotein displacement, its mechanism has not been resolved for any human DNA helicase. Here, we present a quantitative model for the translocation of a monomeric form of BLM along ssDNA. We show that BLM performs translocation at a low adenosine triphosphate (ATP) coupling ratio (1 ATP consumed/1 nucleotide traveled) and moderate processivity (with a mean number of 50 nucleotides traveled in a single run). We also show that the rate-limiting step of the translocation cycle is a transition between two ADP-bound enzyme states. Via opening of the helicase core, this structural change may drive the stepping of BLM along the DNA track by a directed inchworm mechanism. The data also support the conclusion that BLM performs double-stranded DNA unwinding by fully active duplex destabilization

A genom-integritás megőrzésének molekuláris mechanizmusai = Molecular mechanisms of genome maintenance

A projekt során a homológ rekombináción alapuló DNS-hibajavításban kulcsszerepet játszó DNS-helikáz enzimek (humán Bloom-szindróma (BLM) és bakteriális RecQ helikázok) működésmódjait derítettük fel. A RecQ-családba tartozó DNS-helikázok hibás működése emberben magas rák-prediszpozíciót és felgyorsult öregedést eredményez. Kidolgoztunk egy, a nukleinsavak mentén mozgó motorenzimek (pl. DNS-helikázok) legfontosabb működési sajátságainak meghatározására alkalmas analitikai eljárást. E módszer segítségével megalkottuk a RecQ és BLM enzimek mechanokémiai működésének részletes kvantitatív modelljeit. Felfedeztük, hogy a BLM helikáz a monomer és oligomer állapotok közötti dinamikus váltásra képes attól függően, hogy a rekombináció során milyen DNS-szerkezetekkel kerül kölcsönhatásba. Felderítettük a BLM enzim különböző szerkezeti elemeinek a DNS-hibajavítás során mutatott aktivitásokban betöltött szerepeit is. Mivel a BLM helikáz a genomépség fenntartásának elengedhetetlen szereplője, felfedezéseink hozzájárulnak a rákos megbetegedésekhez vezető élettani folyamatok pontosabb megértéséhez és befolyásolásának lehetőségéhez. | This project was aimed at the elucidation of the mechanisms of action of DNA helicases during homologous recombination-based DNA repair processes. DNA helicases essentially contribute to the repair of DNA lesions and error-free transmission of genetic information. Their malfunctions cause high cancer predisposition and accelerated ageing. We investigated the mechanisms of action of the human BLM (Bloom syndrome) and E. coli RecQ helicases. We developed an analytical method suitable for the determination of all key functional parameters of nucleic-acid based motor enzymes, including helicases. Based on this method, we provided quantitative mechanochemical models for the translocation of BLM and RecQ helicases along DNA. We also discovered that BLM dynamically switches between monomeric and oligomeric assembly states during various stages of recombination, depending on the DNA structure encountered. Furthermore, we identified the roles of different domains of BLM helicase in its enzymatic activities exerted during DNA repair processes. As BLM helicase is a key player in the maintenance of the integrity of genetic material, the obtained new insights will aid future efforts to understand and control biological processes that lead to a range of cancerous diseases

A genom-integritás megőrzésének molekuláris mechanizmusai = Molecular mechanisms of genome maintenance

A projekt során végzett munka az NNF78783 sz. projekt egyéves kiterjesztését képviseli. A projekt célja azoknak a mechanizmusoknak a felderítése volt, amelyek révén a RecQ-családbeli helikáz enzimek hozzájárulnak a genom épségének fenntartásához. A munka során az E. coli RecQ és a humán Bloom-szindróma (BLM) és RecQ5β helikázok működéseit vizsgáltuk. Meghatároztuk a Rad51 rekombináz nukleoprotein szálak felépülésének és lebomlásának mechanizmusát, illetve a BLM és RecQ5β helikázok hatását e folyamatokra. Felderítettük azt is, hogy az egyszálú DNS-kötő (SSB) fehérjével létesített kölcsönhatás hogyan befolyásolja a RecQ helikáz enzimatikus és DNS-átalakító aktivitásait. E mechanizmusok központi jelentőségűek a homológ rekombináció elindításában és minőség-ellenőrzésében, és ezáltal a genom épségének fenntartásában. | The work performed in this project was a 1-year continuation of project NNF78783. The overall goal of the project was to elucidate the molecular mechanisms by which RecQ-family DNA helicases contribute to the maintenance of genome integrity. We investigated the action of E. coli RecQ and human Bloom’s syndrome (BLM) and RecQ5β helicases. We determined the mechanism of Rad51 recombinase nucleoprotein filament formation and disassembly, and the influence of BLM and RecQ5β helicases on these processes. We also determined how the interaction with single-stranded DNA binding (SSB) protein influences the enzymatic and DNA-restructuring activities of RecQ helicase. These mechanisms are crucial for the initiation and quality control of homologous recombination, a key process maintaining the integrity of the genome

Mechanism of RecQ helicase mechanoenzymatic coupling reveals that the DNA interactions of the ADP-bound enzyme control translocation run terminations

ABSTRACT The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom's syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning

The mechanism of the reverse recovery-step, phosphate release, and actin activation of Dictyostelium myosin II.

The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation

Direct myosin-2 inhibition enhances cerebral perfusion resulting in functional improvement after ischemic stroke

Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries. Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation. Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry. Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus.Published versio

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom’s syndrome helicase for efficient disruption of D-loops

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM’s multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM’s interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms

Streamlined determination of processive run length and mechanochemical coupling of nucleic acid motor activities

Quantitative determination of enzymatic rates, processivity and mechanochemical coupling is a key aspect in characterizing nucleotide triphosphate (NTP)-driven nucleic acid motor enzymes, for both basic research and technological applications. Here, we present a streamlined analytical method suitable for the determination of all key functional parameters based on measurement of NTP hydrolysis during interaction of motor enzymes with the nucleic acid track. The proposed method utilizes features of kinetic time courses of NTP hydrolysis that have not been addressed in previous analyses, and also accounts for the effect of protein traps used in kinetic experiments on processivity. This analysis is suitable for rapid and precise assessment of the effects of mutations, physical conditions, binding partners and other effectors on the functioning of translocases, helicases, polymerases and other NTP-consuming processive nucleic acid motors