Molecular Mechanism of Sequence-Specific Termination of Lentiviral Replication

Document Type

Article

Publication Date

10-1-2001

Publication Title

Biochemistry

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.

Comments

This work was supported through NIH Grant GM52263.

DOI

10.1021/bi010354i

Volume

40

Issue

40

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