Molecular mechanism of sequence-specific termination of lentiviral replication.
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 K(d) 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 T(6)-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 T(6)-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.
Berdis
A J
AJ
USA Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA. ajb15@po.cwru.edu
Stetor
S R
SR
LeGrice
S F
SF
Barkley
M D
MD
D001483:Base Sequence; D002384:Catalysis; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase