title: Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2.
abstract: Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV).
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors: Etin-Diah
authors: ED
authors: Permanasari
authors: Kiyoshi
authors: K
authors: Yasukawa
authors: Shigenori
authors: S
authors: Kanaya
mesh_terms: D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000254:growth & development; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D011993:Recombinant Fusion Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D012260:Ribonucleases