Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et al. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].oligo [d(T)] as template-primer. We find that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r(A)].oligo[d(T)] replication system. This suggests that 3'-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3'-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3'-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3'-azido-dTTP as substrate revealed that the Michaelis constant is 3 microM. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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http://dx.doi.org/10.1021/bi00467a003 | DOI Listing |
Sci Rep
January 2025
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Olomouc, Czech Republic.
Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication.
View Article and Find Full Text PDFJ Thromb Haemost
January 2025
Dalla Lana School of Public Health, University of Toronto, Toronto, Canada; Office of the Vice-Principal of Research and Innovation, University of Toronto Mississauga, Mississauga, Canada. Electronic address:
Background: Whether to stop oral anticoagulants after a first unprovoked venous thromboembolism (VTE) is challenging, partially due to an intriguingly higher risk of VTE recurrence (rVTE) in men after therapy discontinuation. DNA methylation (DNAm) differences between men and women might underly this sex-biased rVTE risk difference.
Aim: To investigate sex-specific associations between DNAm at cytosine-phosphate-guanine (CpG) sites and rVTE.
Forensic Sci Int Genet
January 2025
Bundeskriminalamt, Wiesbaden, Germany; International Commission on Missing Persons, The Hague, The Netherlands.
The ReAct (Recovery, Activity) project is an ENFSI (European Network of Forensic Science Institutes) supported initiative comprising a large consortium of laboratories. Here, the results from more than 23 laboratories are presented. The primary purpose was to design experiments simulating typical casework circumstances; collect data and to implement Bayesian networks to assess the value (i.
View Article and Find Full Text PDFDNA Repair (Amst)
January 2025
School of Molecular Biosciences, University of Glasgow, Glasgow G12 8QQ, Scotland. Electronic address:
Ubiquitin-specific protease 1 (USP1) is the founding member of the family of cysteine proteases that catalyse hydrolysis of the isopeptide bond between ubiquitin and targets. USP1 is often overexpressed in various cancers, and expression levels correlate with poor prognosis. USP1 and its partner USP1-associated Factor 1 (UAF1) are required for deubiquitinating monoubiquitin signals in DNA interstrand crosslink repair, and in Translesion synthesis, among others, and both proteins are subject to multiple regulations themselves.
View Article and Find Full Text PDFJ Pharm Biomed Anal
January 2025
College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea; Department of Global Innovative Drugs, The Graduate School of Chung-Ang University, Seoul 06974, Republic of Korea. Electronic address:
Semaglutide and liraglutide are long-acting glucagon-like peptide-1 receptor agonists used to treat type-2 diabetes and obesity. Recent advances in peptide synthesis and analytical technologies have enabled the development of synthetic generic peptide for reference listed drugs (RLD) originating from recombinant DNA (rDNA) technology. Since the original semaglutide and liraglutide were produced through rDNA technology, there has been great interest in developing their synthetic peptides as generic versions of the original drugs.
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