Molecular Mechanism of Sequence-Specific Termination of Lentiviral Replication†
Identifieur interne : 003464 ( Main/Exploration ); précédent : 003463; suivant : 003465Molecular Mechanism of Sequence-Specific Termination of Lentiviral Replication†
Auteurs : Anthony J. Berdis [États-Unis] ; Scott R. Stetor [États-Unis] ; Stuart F. J. Legrice [États-Unis] ; Mary D. Barkley [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 2001.
Abstract
The central termination sequence (CTS) terminates (+) strand DNA synthesis in certain lentiviruses. The molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), was evaluated by pre-steady-state kinetic techniques. Time courses in nucleotide incorporation using several DNA substrates were biphasic, consistent with release of enzyme from extended DNA being the rate-limiting step for turnover. While the burst amplitude reflecting the amount of functional RT−DNA complex was sequence-dependent, rate constants for initial product formation were not. Filter binding assays indicate the Kd for CTS-containing substrate is only 2-fold higher than a random DNA and cannot account entirely for the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymerization is unaffected by the T6-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproductive enzyme−nucleic acid complex formation after incorporation of four to five adenosines of a T6-tract within the CTS. During strand displacement synthesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement synthesis decreases 2-fold while the rate of nonproductive enzyme−nucleic acid complex formation is identical in the absence or presence of the displacement strand. These results have allowed us to develop a model for CTS-induced termination of (+) strand synthesis.
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DOI: 10.1021/bi010354i
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The molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), was evaluated by pre-steady-state kinetic techniques. Time courses in nucleotide incorporation using several DNA substrates were biphasic, consistent with release of enzyme from extended DNA being the rate-limiting step for turnover. While the burst amplitude reflecting the amount of functional RT−DNA complex was sequence-dependent, rate constants for initial product formation were not. Filter binding assays indicate the Kd for CTS-containing substrate is only 2-fold higher than a random DNA and cannot account entirely for the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymerization is unaffected by the T6-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproductive enzyme−nucleic acid complex formation after incorporation of four to five adenosines of a T6-tract within the CTS. During strand displacement synthesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement synthesis decreases 2-fold while the rate of nonproductive enzyme−nucleic acid complex formation is identical in the absence or presence of the displacement strand. These results have allowed us to develop a model for CTS-induced termination of (+) strand synthesis.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li><li>Ohio</li></region></list><tree><country name="États-Unis"><region name="Ohio"><name sortKey="Berdis, Anthony J" sort="Berdis, Anthony J" uniqKey="Berdis A" first="Anthony J." last="Berdis">Anthony J. Berdis</name></region><name sortKey="Barkley, Mary D" sort="Barkley, Mary D" uniqKey="Barkley M" first="Mary D." last="Barkley">Mary D. Barkley</name><name sortKey="Berdis, Anthony J" sort="Berdis, Anthony J" uniqKey="Berdis A" first="Anthony J." last="Berdis">Anthony J. Berdis</name><name sortKey="Legrice, Stuart F J" sort="Legrice, Stuart F J" uniqKey="Legrice S" first="Stuart F. J." last="Legrice">Stuart F. J. Legrice</name><name sortKey="Stetor, Scott R" sort="Stetor, Scott R" uniqKey="Stetor S" first="Scott R." last="Stetor">Scott R. Stetor</name><name sortKey="Stetor, Scott R" sort="Stetor, Scott R" uniqKey="Stetor S" first="Scott R." last="Stetor">Scott R. Stetor</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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