Preferred sequences within a defined cleavage window specify DNA 3' end-directed cleavages by retroviral RNases H.
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.
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Sharon J
SJ
Schultz
Miaohua
M
Zhang
James J
JJ
Champoux
D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics*; D016914:Ribonuclease H / Q000737:chemistry*
D004279:DNA, Viral; D012367:RNA, Viral; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI