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Journal of Virology, September 2006, p. 8379-8389, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00750-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain

Integrated Program in Cellular, Molecular and Biophysical Studies,¹ Howard Hughes Medical Institute,² Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032³

Received 12 April 2006/ Accepted 17 May 2006

A crystallographic study of the Moloney murine leukemia virus (Mo-MLV) RNase H domain was performed to provide information about its structure and mechanism of action. These efforts resulted in the crystallization of a mutant Mo-MLV RNase H lacking the putative helix C (C). The 1.6-Å resolution structure resembles the known structures of the human immunodeficiency virus type 1 (HIV-1) and Escherichia coli RNase H. The structure revealed the coordination of a magnesium ion within the catalytic core comprised of the highly conserved acidic residues D524, E562, and D583. Surface charge mapping of the Mo-MLV structure revealed a high density of basic charges on one side of the enzyme. Using a model of the Mo-MLV structure superimposed upon a structure of HIV-1 reverse transcriptase bound to an RNA/DNA hybrid substrate, Mo-MLV RNase H secondary structures and individual amino acids were examined for their potential roles in binding substrate. Identified regions included Mo-MLV RNase H ß1-ß2, A, and B and residues from B to D and its following loop. Most of the identified substrate-binding residues corresponded with residues directly binding nucleotides in an RNase H from Bacillus halodurans as observed in a cocrystal structure with RNA/DNA. Finally, superimposition of RNases H of Mo-MLV, E. coli, and HIV-1 revealed that a loop of the HIV-1 connection domain resides within the same region of the Mo-MLV and E. coli C-helix. The HIV-1 connection domain may serve to recognize and bind the RNA/DNA substrate major groove.

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