Reduced dNTP interaction of human immunodeficiency virus type 1 reverse transcriptase promotes strand transfer

We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.     

A mutation in the rnh-locus of Escherichia coli affects the structural gene for RNase H. Examination of the mutant and wild type protein

Ribonuclease H (RNase H, EC 3.1.26.4) was purified to homogeneity from Escherichia coli wild type strain KS 351 and the RNase H mutant strain FB 2. The specific activity of the wild type enzyme was 43,200 units/mg, while that of the mutant enzyme was 3,430 units/mg, less than 8% of the wild type activity. Isoelectric focusing also revealed differences in the protein from mutant and wild type. The activity of the wild type enzyme was separated into two peaks with isoelectric points of 9.6 and 9.0. In contrast, the activity of the mutant enzyme focused in a single peak with a pI of 9.4. These results indicate that the mutation in the FB2 strain affects the structural gene for RNase H. The molecular weight of both enzymes was determined by gel filtration as well as NaDodSO4-polyacrylamide gel electrophoresis and found to be identical. Both enzymes are very sensitive to increased temperatures and show indistinguishable rates of inactivation. The basis for the heterogeneity of the isoelectric point and the altered activity of the mutant enzyme is still unknown.     

Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.