Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: Implications for primer binding

Journal Article · Tue Nov 05 00:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00108a004· OSTI ID:5559313

Sobol, R W; Suhadolnik, R J ^[1]; Kumar, A; Lee, Byeong Jae; Hatfield, D L; Wilson, S H ^[2]

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1. Temple Univ., Philadelphia, PA (United States)
2. National Inst. of Health, Bethesda, MD (United States)

Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T){sub 8} and pd(T){sub 16} as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers indicate that pd(T){sub n} binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T){sub n} binding to RT has a large nonelectrostatic component. The site of UV cross-linking of pd(T){sub 16} was localized to the NH{sub 2}-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. The results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195{approximately}300. This region could be either a single-stranded template or single-stranded primer binding site; however, the authors have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T){sub 16} binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

• OSTI ID: 5559313
• Journal Information: Biochemistry; (United States), Vol. 30:44; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
BIOLOGICAL RADIATION EFFECTS
RNA
TRANSCRIPTION
BIOCHEMICAL REACTION KINETICS
CROSS-LINKING
ESCHERICHIA COLI
EXTREME ULTRAVIOLET RADIATION
PHOSPHORUS 31
BACTERIA
BIOLOGICAL EFFECTS
CHEMICAL REACTIONS
ELECTROMAGNETIC RADIATION
ENZYMES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
POLYMERIZATION
PROTEINS
RADIATION EFFECTS
RADIATIONS
REACTION KINETICS
STABLE ISOTOPES
TRANSFERASES
ULTRAVIOLET RADIATION
550201* - Biochemistry- Tracer Techniques