Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase.
The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
Cirino
N M
NM
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Cameron
C E
CE
Smith
J S
JS
Rausch
J W
JW
Roth
M J
MJ
Benkovic
S J
SJ
Le Grice
S F
SF
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology*; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000254:growth & development; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D012313:RNA; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D016914:Ribonuclease H / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; C034477:RNA primers; D012367:RNA, Viral; D011994:Recombinant Proteins; D008345:Manganese; D012313:RNA; D012343:RNA, Transfer; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H; D008274:Magnesium