Sj. Schultz et Jj. Champoux, RNASE-H DOMAIN OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASERETAINS ACTIVITY BUT REQUIRES THE POLYMERASE DOMAIN FOR SPECIFICITY, Journal of virology, 70(12), 1996, pp. 8630-8638
The reverse transcriptase-associated RNase H activity of Moloney murin
e leukemia virus specifically cleaves within the polypurine tract regi
on of the viral genome to generate the primer for plus-strand DNA synt
hesis and removes the tRNA primer after minus-strand initiation bg pre
ferentially cleaving the RNA one nucleotide before the RNA-DNA junctio
n. Moreover, the enzyme is unable to cleave the extended tRNA substrat
e at the RNA-DNA junction even at high enzyme concentrations. The RNas
e H domain of the reverse transcriptase was expressed as a glutathione
S-transferase fusion protein and purified from Escherichia coli extra
cts, Following removal of the glutathione S-transferase portion of the
protein, the specificity of the isolated RNase H domain was determine
d in the plus-strand primer reaction and in the tRNA primer removal re
action, Although the isolated domain lacked specificity in both cases,
it was still unable to cleave the tRNA substrate precisely at the RNA
-DNA junction. Specificity in both cases could be restored by adding b
ack a truncated form of Moloney murine leukemia virus reverse transcri
ptase lacking the RNase a domain, These results implicate the polymera
se domain as a specificity determinant for the RNase H activity of rev
erse transcriptase. The isolated RNase H domain had higher activity in
the presence of Mn2+ than in the presence of Mg2+, but neither the RN
ase H domain alone nor the RNase H domain coupled to the polymerase do
main in wild-type protein exhibited the normal cleavage specificities
in the presence of the nonphysiological divalent cation.