Abstract
RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.
Highlights
RNA/DNA substrate [11, 15]
This unexpected mode derived from H is considered a nonhumanimmunodeficiency virus (HIV), manifested by different reverse transcriptase (RT) murine leukemia virus (MMuLV), and AMV RT on homologous of action of RNase Hat the 6’ end of the genomic RNA and heterologous genomes, (ii) what is the minimal hybrid should betaken in consideration in studieofs the first size recognized by the RNase H of the three enzymes, and template switch
In thisstudy we have comparedthe specificity of the RNase labeled at the 5’ end. b, E. coli RNase H activity on the residual protected hybrid that remained after reaction with HIV RT
Summary
SP6 RNA polymerase in the presence of [LY-~’P]CTPF.ollowing RNA synthesis, the products were treated with 2 units of DNase I, extracted with phenol chloroform, and ethanol precipitated. The 18, 47, and 65 nucleotide oligomers were hybridized to the a-32P-labeled RNA by first incubating at 65 “Cfor 5 min in buffer containing 30%. Cleavage formamide, 40 mM Tris-HC1, pH 7.4, and 1 mM EDTA and of substrate B generated distinct 3‘ and 5‘ end RNA products adding NaCl to 0.3 M and incubating an additional 90 min at 42 “C. The 13 an2d8 deoxynucleotideoligomerswere annealed to the 5’ end of the RNA template by first incubating a t 65 “Cfor 5 min in 0.12 M sodium citrate, pH 7.0, and adding NaCl to 1.12 M and incubating for 3.5 h at 30 “C.The hybrids were ethanol precipitated, dissolved in H20, and used in the RNase H activity assay, as described. The 5’ end RNA products obtained after RNase H digestion were resolved in a 20% sequencing gel.As can be seen in Fig.2a, each of the three
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