Whiting S H, Champoux J J
School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
J Mol Biol. 1998 May 8;278(3):559-77. doi: 10.1006/jmbi.1998.1720.
Previous results indicated that Moloney murine leukemia virus reverse transcriptase is capable of extensive synthesis under conditions where it must simultaneously displace a downstream non-template DNA strand. To investigate more fully the mechanistic basis for displacement synthesis and to characterize the activity with natural viral templates, displacement and non-displacement synthesis were compared under a variety of conditions using the viral long terminal repeat plus strand as the template. Although the rates of both displacement and non-displacement synthesis varied regionally over the template, on the average, displacement synthesis was slower by a factor of approximately 3 to 4. Surprisingly, with one particular primer situated downstream of the tRNA primer binding site, displacement synthesis was found to be at least tenfold more processive than non-displacement synthesis, approaching a value of 500 nucleotides. The sequence features associated with pausing during the two modes of synthesis are different in both nucleotide preference and position relative to the enzyme, suggesting that the enzyme contacts the DNA differently under the two modes of synthesis. It was found that pausing during displacement synthesis did not reflect those local regions of DNA with a predicted high degree of thermal stability. Moreover, the very similar effects of temperature on the rates of displacement and non-displacement synthesis make unlikely a strictly passive mechanism of displacement synthesis whereby breathing of the downstream duplex is sufficient for advancement of the polymerase. Together, these results suggest a mechanism of displacement synthesis in which reverse transcriptase actively participates in the process of strand separation in front of the translocating polymerase.
先前的结果表明，莫洛尼鼠白血病病毒逆转录酶在必须同时置换下游非模板DNA链的条件下能够进行大量合成。为了更全面地研究置换合成的机制基础，并表征其在天然病毒模板上的活性，使用病毒长末端重复序列正链作为模板，在各种条件下比较了置换合成和非置换合成。尽管置换合成和非置换合成的速率在模板上各区域有所不同，但平均而言，置换合成慢约3至4倍。令人惊讶的是，对于位于tRNA引物结合位点下游的一种特定引物，发现置换合成的持续合成能力比非置换合成至少高十倍，接近500个核苷酸的值。两种合成模式下与暂停相关的序列特征在核苷酸偏好和相对于酶的位置上均不同，这表明酶在两种合成模式下与DNA的接触方式不同。研究发现，置换合成过程中的暂停并不反映那些预测具有高度热稳定性的DNA局部区域。此外，温度对置换合成和非置换合成速率的影响非常相似，这使得置换合成不太可能是一种严格的被动机制，即下游双链的解链足以使聚合酶前进。总之，这些结果提示了一种置换合成机制，其中逆转录酶在易位聚合酶前方的链分离过程中积极参与。
