RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome.
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.
Ben-Artzi
H
H
BioTechnology General Ltd., Kiryat Weizmann, Rehovot, Israel.
Zeelon
E
E
Amit
B
B
Wortzel
A
A
Gorecki
M
M
Panet
A
A
D009189:Avian Myeloblastosis Virus / Q000201:enzymology / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism; D016679:Genome, Viral; D006678:HIV / Q000201:enzymology / Q000235:genetics*; D054303:HIV Reverse Transcriptase; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity
D004279:DNA, Viral; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H