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Volume 272, Number 35, Issue of August 29, 1997 pp. 22023-22029
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Isolated RNase H Domain of Murine Leukemia Virus Reverse Transcriptase
RETENTION OF ACTIVITY WITH CONCOMITANT LOSS OF SPECIFICITY

(Received for publication, October 21, 1996, and in revised form, June 25, 1997)

From the Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn²⁺ as the cation required for activity, and has association rate (K_A) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNA^Pro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in K_A, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.

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