Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90.
Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.
Basu
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Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark 07103.
Basu
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Modak
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D001483:Base Sequence; D001577:Benzophenones; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000032:analysis* / Q000502:physiology; D017931:DNA Primers / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008301:Maleimides; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013698:Templates, Genetic; D014357:Trypsin; D014466:Ultraviolet Rays
D001577:Benzophenones; D003432:Cross-Linking Reagents; D017931:DNA Primers; D008301:Maleimides; D018894:Reverse Transcriptase Inhibitors; D013438:Sulfhydryl Compounds; C046109:benzophenone-4-maleimide; D012194:RNA-Directed DNA Polymerase; D014357:Trypsin; D003545:Cysteine