During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

• MeSH
• DNA primery metabolismus   MeSH
• DNA-primasa * metabolismus   MeSH
• DNA genetika   MeSH
• katalytická doména MeSH
• replikace DNA * 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
• DNA primery MeSH
• DNA-primasa * MeSH
• DNA MeSH

Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• hořčík chemie   MeSH
• infekce virem zika genetika   virologie   MeSH
• konformace proteinů účinky léků   MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleosidy chemie   MeSH
• nukleotidy chemie   MeSH
• polyfosfáty chemie   MeSH
• replikace viru genetika   MeSH
• RNA-dependentní RNA-polymerasa chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• simulace molekulového dockingu MeSH
• virové nestrukturální proteiny chemie   genetika   MeSH
• virus zika chemie   genetika   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• hořčík MeSH
• NITD008 MeSH Prohlížeč
• nukleosidy MeSH
• nukleotidy MeSH
• polyfosfáty MeSH
• RNA-dependentní RNA-polymerasa MeSH
• RNA MeSH
• triphosphoric acid MeSH Prohlížeč
• virové nestrukturální proteiny MeSH

7-Deazapurine (pyrrolo[2,3-d]pyrimidine) nucleosides are important analogues of biogenic purine nucleosides with diverse biological activities. Replacement of the N7 atom with a carbon atom makes the five-membered ring more electron rich and brings a possibility of attaching additional substituents at the C7 position. This often leads to derivatives with increased base-pairing in DNA or RNA or better binding to enzymes. Several types of 7-deazapurine nucleosides with potent cytostatic or cytotoxic effects have been identified. The most promising are 7-hetaryl-7-deazaadenosines, which are activated in cancer cells by phosphorylation and get incorporated both to RNA (causing inhibition of proteosynthesis) and to DNA (causing DNA damage). Mechanism of action of other types of cytostatic nucleosides, 6-hetaryl-7-deazapurine and thieno-fused deazapurine ribonucleosides, is not yet known. Many 7-deazaadenosine derivatives are potent inhibitors of adenosine kinases. Many types of sugar-modified derivatives of 7-deazapurine nucleosides are also strong antivirals. Most important are 2'-C-methylribo- or 2'-C-methyl-2'-fluororibonucleosides with anti-HCV activities (several compounds underwent clinical trials). Some underexplored areas of potential interest are also outlined.

• Klíčová slova
• antivirals, cytostatics, deazapurines, nucleosides, nucleotides,
• MeSH
• antivirové látky chemická syntéza   chemie   farmakologie   MeSH
• buňky A549 MeSH
• buňky Hep G2 MeSH
• HeLa buňky MeSH
• lidé MeSH
• nukleosidy chemická syntéza   chemie   farmakologie   MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   MeSH
• puriny chemie   MeSH
• racionální návrh léčiv MeSH
• screeningové testy protinádorových léčiv MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 7-deazapurine MeSH Prohlížeč
• antivirové látky MeSH
• nukleosidy MeSH
• protinádorové látky MeSH
• puriny MeSH

Three types of sugar modified pyrimido[4,5-b]indole nucleosides (2'-deoxy-2'-fluororibo-, 2'-deoxy-2'-fluoroarabino- and arabinonucleosides) were synthesized by glycosylation of 4,6-dichloropyrimido[4,5-b]indole followed by modification of sugar moiety and introduction of substituents into position 4 by cross-coupling reactions or nucleophilic substitutions. Some 2'-fluororibo- and 2'-fluoroarabinonucleosides displayed interesting anti-HCV activities (IC50 = 1.6-20 μM) and the latter compounds also some anti-dengue activities (IC50 = 10.8-40 μM).

• Publikační typ
• časopisecké články MeSH