RESUMEN
The human N-glycosylases SMUG1 and MBD4 catalyze the removal of uracil residues from DNA resulting from cytosine deamination or replication errors. For polymorphic variants of SMUG1 (G90C, P240H, N244S, N248Y) and the MBD4^(cat) catalytic domain (S470L, G507S, R512W, H557D), the structures of enzyme-substrate complexes were obtained by molecular dynamic simulation. It was experimentally found that the SNP variants of SMUG1, N244S and N248Y, had increased catalytic activity compared to the wild-type enzyme, probably due to the acceleration of the dissociation of the enzyme-product complex and an increase in the enzyme turnover rate. All other SNP variants of SMUG1 (G90C, P240H) and MBD4^(cat), in which amino acid substitutions disrupted the substrate binding region and/or active site, had significantly lower catalytic activity than the wild-type enzymes.
Asunto(s)
Reparación del ADN , Uracil-ADN Glicosidasa , ADN , Daño del ADN , Endodesoxirribonucleasas , Humanos , Uracilo , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismoRESUMEN
Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme's tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.
Asunto(s)
Arginina/química , Dominio Catalítico , Daño del ADN , Reparación del ADN , Histidina/química , Uracil-ADN Glicosidasa/química , Uracil-ADN Glicosidasa/metabolismo , Secuencia de Aminoácidos , Humanos , Cinética , Simulación de Dinámica Molecular , Homología de Secuencia , Especificidad por Sustrato , Uracilo/metabolismo , Uracil-ADN Glicosidasa/genéticaRESUMEN
Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Sondas de ADN/química , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Pruebas de Enzimas/métodos , Colorantes Fluorescentes/química , Extractos Celulares , Células Cultivadas , Enzimas Reparadoras del ADN/genética , HumanosRESUMEN
Surgical treatment of stenosis of the left main coronary artery (LMCA) is a special problem, a number of questions of surgical strategy for this lesion still remain unsolved. The question of direct plasty dilatation of LMCA has not lost its actuality. Operations were made on 6 patients in whom direct plasty dilatation of LMCA with a flap from autopericardium was performed for local, not lengthy constriction which was located in the initial and middle part of LMCA. The operations were made under conditions of extracorporeal circulation, moderate general hypothermia (about 32 degrees C), pharmacohypothermic cardioplagia with external cooling of the heart. In all cases there was good restoration of cardiac activity and the absence of myocardium ischemia at the postoperative period. The restoration of the anatomical lumen of LMCA by this method facilitated sufficient blood flow along LMCA. Five years later all the patients are alive, one patient has external angina of the 11 functional class.