title: Substitution of Asp114 or Arg116 in the fingers domain of moloney murine leukemia virus reverse transcriptase affects interactions with the template-primer resulting in decreased processivity.
abstract: Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.
authors: Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
authors: J
authors: J
authors: Gu
authors: R A
authors: RA
authors: Villanueva
authors: C S
authors: CS
authors: Snyder
authors: M J
authors: MJ
authors: Roth
authors: M M
authors: MM
authors: Georgiadis
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011533:Proviruses / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins / Q000096:biosynthesis; D014771:Virion / Q000201:enzymology / Q000302:isolation & purification / Q000502:physiology; D014779:Virus Replication
chemical_list: C034477:RNA primers; D012333:RNA, Messenger; D014764:Viral Proteins; D001224:Aspartic Acid; D012313:RNA; D004247:DNA; D001120:Arginine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H