Valine of the YVDD motif of Moloney murine leukemia virus reverse transcriptase: Role in the fidelity of DNA synthesis

The YXDD motif is highly conserved in the reverse transcriptase family. The variable X residue is occupied by valine and methionine in MuLV RT and HIV -1 RT, respectively. Previous studies have shown that Tyr 222, the Y residu e of the YXDD motif in MuLV RT, constitutes a major component of the fideli ty center of the enzyme [Kaushik, N., Singh, K., Alluru, I., and Modak, M. J. (1999) Biochemistry 38, 2617-2627], In this work, we present evidence th at reverse transcriptases containing valine in the "X" position of the YXDD motif generally catalyze DNA synthesis with greater fidelity than those co ntaining methionine or alanine. In the MuLV RT system, the two mutants V223 M and V223A exhibited an overall reduced fidelity of DNA synthesis, specifi cally for RNA-templated reactions. Further analysis revealed that these mut ants exhibit a higher efficiency of misinsertion on MS2 RNA than the wild-t ype enzyme for every mispair tested. However, unlike HIV-1 RT, the insensit ivity of the wild-type MuLV RT to all four ddNTPs remained unchanged by mut ation of V223 to Met or Ala. A 3D molecular model of the ternary complex of MuLV RT, template primer, and dNTP suggests that Val 223 along with its ne ighboring Tyr 222 stabilizes the substrate binding pocket via hydrophobic i nteractions with the dNTP substrate and template-primer.