Chemical approaches to the elucidation of template biosynthesis: study of replication and reverse transcription

Abstract

Chemical approaches to the elucidation of DNA tem plate biosynthesis using DNA polymerases and reverse tran scriptases, predominantly DNA polymerase I from E. coli and reverse transcriptase from HIV-1, are considered. Special atten tion is paid to the comparison of chemical modification and X-ray structural analysis data as well as to quantitative aspects of the interaction of DNA polymerases with templates, primers, and nucleoside 5 H -triphosphates. New versions of a catalytically competent labelling method developed in the course of investigation of these enzymes are described. The data obtained in the analysis of replication complexes using primer analogues with photoreactive groups are presented. The bibliography includes 98 references.

Synthesis of Base-Substituted dUTP Analogues Carrying a Photoreactive Group and Their Application to Study Human Replication Protein A

“…Base-substituted analogues of dNTPs carrying arylazido groups provide new tools for photoaffinity labeling of the proteins interacting with nucleic acids. A number of aryl azides were used to obtain base-substituted photoreactive analogues of dUTP (Knorre et al, 1998). Synthesis of a photoreactive primer-template using NAB-4-dUTP as a substrate of various polymerases was reported earlier, and this primer-template was used to study RPA interaction with DNA duplex (Lavrik et al, 1998).…”

“…In particular, nucleotides carrying aryl azides have been widely used to investigate protein-nucleic acid and nucleic acid-nucleic acid interactions. Analogues of dUTP 1 (UTP) with a bulky group conjugated at the C5 on the pyrimidine ring are substrates for a number of DNA and RNA polymerases (Knorre et al, 1998), which makes them useful tools to study interactions between these substrates and components of the DNA replication machinery. Thus, the insight into the assembly of the replication (transcription) machinery might be deepened by the use of these photoreactive analogues.…”

Photoactivatable Analogues of the Initiating Substrates of RNA Polymerase II Based on Aryl Azide Derivatives of NTP γ-Amidophosphate: Synthesis and Chemical and Photochemical Reactions of Functional Groups

“…The photoactivatable groups within ATP derivatives had previously been widely used as components of affinity reagents in the study of various systems, including the template biosynthesis [1,30]. Nevertheless, the photolysis process in aqueous solution as well as the products of their photoconversion have been remained poorly investigated up to now despite the fact that they could certainly affect the results obtained at the biopolymer modification [11].…”