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J. Biol. Chem., Vol. 281, Issue 4, 1943-1955, January 27, 2006

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Sequence, Distance, and Accessibility Are Determinants of 5'-End-directed Cleavages by Retroviral RNases H^*

From the Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195

The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5'-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5'-end. Previously, the distance from the RNA 5'-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5'-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5'-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the +1 position and C or G at the –2 position, and additionally for HIV-1, A is disfavored at the –4 position. For both enzymes, 5'-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5'-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5'-end, it was found that the extent of 5'-end-directed cleavages observed in substrates containing a free recessed RNA 5'-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5'-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5'-end.

Received for publication, September 26, 2005 , and in revised form, November 11, 2005.

^* This work was supported by National Institutes of Health Grant CA51605. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology, Box 357242, School of Medicine, University of Washington, Seattle, WA 98195-7242. Tel.: 206-543-8574; Fax: 206-543-8297; E-mail: champoux{at}u.washington.edu.

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