Interaction of the Ty3 reverse transcriptase thumb subdomain with template-primer.
Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.
Bibillo
Arkadiusz
A
Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI, Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Lener
Daniela
D
Tewari
Alok
A
Le Grice
Stuart F J
SF
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D018626:Retroelements; D016914:Ribonuclease H / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry
D017931:DNA Primers; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C496084:reverse transcriptase Ty3, S cerevisiae; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H; D000409:Alanine