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Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase

Title data

Souquet, Manfred ; Restle, Tobias ; Krebs, Ruth ; Le Grice, Stuart F.J. ; Goody, Roger S. ; Wöhrl, Birgitta:
Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase.
In: Biochemistry. Vol. 37 (1998) Issue 35 . - pp. 12144-12152.
ISSN 1520-4995
DOI: https://doi.org/10.1021/bi9731596

Abstract in another language

Homodimeric EIAV p51/51 and heterodimeric EIAV p66/51 reverse transcriptase were purified in order to compare the different modes of DNA synthesis supported by the enzymes. Analysis of the dimerization behavior of the EIAV enzymes indicates that the dimer stability of EIAV reverse transcriptase enzymes is higher than that of their HIV-1 reverse transcriptase counterparts. EIAV p51/51 polymerizes DNA distributively whereas DNA synthesis by EIAV p66/51 is processive. Steady-state and pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation were performed with both enzymes to determine the reasons for the different polymerization behavior. Equilibrium fluorescence titrations demonstrated that the Kd values of EIAV p51/51 for binding of DNA/DNA and DNA/RNA substrates are increased 10-fold and 28-fold, respectively, as compared to EIAV p66/51. Stopped-flow measurements with DNA/DNA show that the increase in the Kd is in part due to a 17. 4-fold higher dissociation rate constant (k₋₁) for EIAV p51/51. Additionally, with EIAV p51/51, kdiss is increased 7-fold for DNA/DNA and 14-fold for DNA/RNA primer/template substrates, respectively. The lack of the RNase H domain in EIAV p51/51 leads to differences in the pre-steady-state kinetics of nucleotide incorporation on DNA/DNA and DNA/RNA templates. The burst of both enzymes is composed of two phases for both substrates, and the values for the corresponding pre-steady-state burst rates, kpol₁ and kpol₂, are similar for both enzymes, implying the formation of identical polymerase active sites. However, the amplitudes of the two phases differ with DNA/DNA templates, indicating a different distribution between two states varying greatly in their kinetic competence

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 20 May 2019 06:22
Last Modified: 20 May 2019 06:22
URI: https://eref.uni-bayreuth.de/id/eprint/48992