Comparison of second-strand transfer requirements and RNase H cleavages catalyzed by human immunodeficiency virus type 1 reverse transcriptase (RT) and E478Q RT.
Truncated tRNA-DNA mimics were examined in an in vitro assay for second-strand transfer during human immunodeficiency virus type 1 (HIV-1) reverse transcription. Strand transfer in this system requires the progressive degradation of the RNA within the 18-mer tRNA-DNA (plus-strand strong stop DNA) intermediate to products approximately 8 nucleotides in length. The ability of the truncated substrates to substitute for directional processing by RNase H or reverse transcriptase (RT) was examined. Using wild-type HIV-1 RT, substrates which truncated the 5' end of the tRNA primer by 6, 9, and 12 nucleotides (Delta6, Delta9, and Delta12, respectively) were recognized by RNase H and resulted in strand transfer. An overlap of 5 nucleotides between the acceptor and newly synthesized DNA template was sufficient for strand transfer. The mutant RT, E478Q correctly catalyzed the initial cleavage of the 18-mer tRNA-DNA mimic in the presence of Mn(2+); however, no directional processing was observed. In contrast, no RNase H activity was observed with the Delta6, Delta9, and Delta12 substrates with E478Q RT in this strand transfer assay. However, when complemented with Escherichia coli RNase H, E478Q RT supported strand transfer with the truncated substrates. E478Q RT did cleave the truncated forms of the substrates, Delta6, Delta9, and Delta12, in a polymerase-independent assay. The size requirements of the substrates which were cleaved by the polymerase-independent RNase H activity of E478Q RT are defined.
Snyder
C S
CS
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Roth
M J
MJ
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D016914:Ribonuclease H / Q000378:metabolism*; D014158:Transcription, Genetic
D004247:DNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H