scholarly journals Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis

2010 ◽  
Vol 150 (3) ◽  
pp. 299-306 ◽  
Author(s):  
Kiyoshi Yasukawa ◽  
Masaki Mizuno ◽  
Atsushi Konishi ◽  
Kuniyo Inouye
Keyword(s):  
Thermal Stability ◽  
Reverse Transcriptase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Leukaemia Virus ◽  
Murine Leukaemia Virus ◽  
Thermal Stability Of ◽  
Moloney Murine Leukaemia Virus

scholarly journals Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90

1993 ◽  
Vol 296 (3) ◽  
pp. 577-583 ◽  
Author(s):  
S Basu ◽  
A Basu ◽  
M J Modak
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Structural Integrity ◽  
Peptide Mapping ◽  
Site Directed Mutagenesis ◽  
Heat Inactivation ◽  
Composition Analysis ◽  
Kinetic Measurements ◽  
Leukaemia Virus ◽  
Murine Leukaemia Virus

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.

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