Preferred Sequences within a Defined Cleavage Window Specify DNA 3′ End-directed Cleavages by Retroviral RNases H

• Published in: The Journal of biological chemistry Vol. 284; no. 47; pp. 32225 - 32238
• Main Authors: Schultz, Sharon J., Zhang, Miaohua, Champoux, James J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.11.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Base Sequence; Catalytic Domain; Crystallography, X-Ray - methods; Data processing; DNA; DNA - genetics; DNA structure; DNA, Viral - genetics; DNA: , Repair, Recombination, and Chromosome Dynamics; HIV-1 - genetics; Human immunodeficiency virus 1; Humans; Molecular Sequence Data; Moloney murine leukemia virus; Moloney murine leukemia virus - genetics; Nucleic Acid Hybridization; Nucleotide sequence; Nucleotides; Primers; Protein Structure, Tertiary; Retroviridae - enzymology; Retroviridae - genetics; Ribonuclease H - chemistry; RNA; RNA, Viral - genetics; RNA-directed DNA polymerase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.043158

The RNase H activity of reverse transcriptase carries out three types of cleavage termed internal, RNA 5′ end-directed, and DNA 3′ end-directed. Given the strong association between the polymerase domain of reverse transcriptase and a DNA 3′ primer terminus, we asked whether the distance from the primer terminus is paramount for positioning DNA 3′ end-directed cleavages or whether preferred sequences and/or a cleavage window are important as they are for RNA 5′ end-directed cleavages. Using the reverse transcriptases of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), we determined the effects of sequence, distance, and substrate end structure on DNA 3′ end-directed cleavages. Utilizing sequence-matched substrates, our analyses showed that DNA 3′ end-directed cleavages share the same sequence preferences as RNA 5′ end-directed cleavages, but the sites must fall in a narrow window between the 15th and 20th nucleotides from the recessed end for HIV-1 reverse transcriptase and between the 17th and 20th nucleotides for M-MuLV. Substrates with an RNA 5′ end recessed by 1 (HIV-1) or 2–3 (M-MuLV) bases on a longer DNA could accommodate both types of end-directed cleavage, but further recession of the RNA 5′ end excluded DNA 3′ end-directed cleavages. For HIV-1 RNase H, the inclusion of the cognate dNTP enhanced DNA 3′ end-directed cleavages at the 17th and 18th nucleotides. These data demonstrate that all three modes of retroviral RNase H cleavage share sequence determinants that may be useful in designing assays to identify inhibitors of retroviral RNases H.