Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.

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

Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by ...

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfe...

Patch cloning method for multiple site-directed and saturation mutagenesis

BACKGROUND Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describe...

Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA.

We have previously described the construction of a mutant of Moloney murine leukemia virus bearing a deletion at the normal site of integration of the viral DNA. We have now recovered a revertant of the virus after abortive infection of mouse cells and have determined the structure of the new virus. The revertant is a recombinant virus containing a 500-base-pair patch of new sequences derived f...