Attenuation of DNA replication by HIV-1 reverse transcriptase near the central termination sequence
Attenuation of DNA replication by HIV-1 reverse transcriptase near the central termination sequence
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DOI：
10.1021/bi048000p
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发表时间：
2005-04-12
期刊：
影响因子：
2.9
通讯作者：
Barkley, MD
中科院分区：
文献类型：
--
作者：
Ignatov, ME;Berdis, AJ;Barkley, MD
先前对马传染性贫血病毒-1（EIAV）逆转录酶（RT）的预稳态动力学研究显示了含有中心终止序列（CTS）的DNA底物对聚合反应的两种影响：在单核苷酸添加实验中爆发幅度的降低和在进行性DNA合成期间终止产物的积累[Berdis，A. J.，Stetor，S. R.，Le Grice，S. F. J.，和Barkley，M. D.（2001）Biochemistry 40，12140-12149]。本研究的HIV RT使用前稳态动力学技术，以评估使用随机序列和含CTS的DNA底物的较低爆发振幅的分子机制。各种因素，包括引物/模板长度，结合方向，和蛋白质浓度，对突发幅度的影响，确定使用随机序列DNA底物。活性RT的百分比随总RT浓度增加，表明RT二聚体的可逆解离是正常底物亚化学计量爆发振幅的原因。凝胶迁移率变动分析证实了这一发现。与EIAV RT一样，HIV RT在含有CTS的DNA底物上显示出比随机序列更低的爆发幅度。通过酶活性和荧光强度监测RT-DNA复合物的解离动力学。随机序列和CTS-含有DNA复合物的双相动力学进行了观察，揭示了两种形式的RT-DNA复合物。提出了一种机制来解释减少含有CTS的DNA的爆发振幅，这与单核苷酸添加和解离实验的结果是一致的。RT-DNA复合物的两种形式可能代表引物/模板在RT上的P-和N-位点之间的分配。
Previous pre-steady-state kinetic studies of equine infectious anemia virus-1 (EIAV) reverse transcriptase (RT) showed two effects of DNA substrates containing the central termination sequence (CTS) on the polymerization reaction: reduction of burst amplitude in single nucleotide addition experiments and accumulation of termination products during processive DNA synthesis [Berdis, A. J., Stetor, S. R., Le Grice, S. F. J., and Barkley, M. D. (2001) Biochemistry 40, 12140-12149]. The present study of HIV RT uses pre-steady-state kinetic techniques to evaluate the molecular mechanisms of the lower burst amplitudes using both random sequence and CTS-containing DNA substrates. The effects of various factors, including primer/template length, binding orientation, and protein concentration, on the burst amplitude were determined using random sequence DNA substrates. The percent active RT increases with total RT concentration, indicating that reversible dissociation of RT dimer is responsible for substoichiometric burst amplitudes with normal substrates. This finding was confirmed by gel mobility shift assays. Like EIAV RT, HIV RT showed lower burst amplitudes on CTS-containing DNA substrates compared to random sequences. The dissociation kinetics of RT-DNA complexes were monitored by enzyme activity and fluorescence. Biphasic kinetics were observed for both random sequence and CTS-containing DNA complexes, revealing two forms of the RT-DNA complex. A mechanism is proposed to account for reduction in burst amplitude of CTS-containing DNA that is consistent with the results of both single nucleotide addition and dissociation experiments. The two forms of the RT-DNA complex may represent partitioning of primer/template between the P- and N-sites on RT for the nucleic acid substrate.