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.
Schultz
Sharon J
SJ
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Zhang
Miaohua
M
Champoux
James J
JJ
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