Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase

• Published in: Biochimica et biophysica acta Vol. 1048; no. 2/3; pp. 139 - 148
• Main Authors: Laquel, P, Sallafranque-Andreola, M, Tarrago-Litvak, L, Castroveijo, M, Litvak, S
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 06.04.1990; North-Holland
• Subjects: AIDS/HIV; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity; Chromatography, Ion Exchange; DNA - biosynthesis; DNA Polymerase II - isolation & purification; DNA Polymerase II - metabolism; DNA Polymerase III - isolation & purification; DNA Polymerase III - metabolism; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; enzyme activity; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HIV - enzymology; isolation; Kinetics; physicochemical properties; reverse transcription; RNA - genetics; RNA, Messenger - genetics; RNA-Directed DNA Polymerase - isolation & purification; RNA-Directed DNA Polymerase - metabolism; Seeds - enzymology; Templates, Genetic; Transcription, Genetic; Transferases; Triticum - enzymology; Triticum - genetics; Triticum aestivum; Vegetals; Purification; DNA polymerase; Radiolabelling; Gel electrophoresis; Enzyme; Retroviridae; Optimization; Ion exchange chromatography; Virus; Enzymatic activity; Affinity chromatography; DNA polymerase α; Substrate specificity; DNA polymerase RNA-dependent; Lentivirinae; Human immunodeficiency virus; Comparative study; Physicochemical properties
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/0167-4781(90)90049-8

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Taffago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase y to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.