Modified Nucleosides as Probes of Replication Complexes

研究了HIV-1RT核糖核酸酶H区识别(+)链DNA合成的多聚尿路引物的结构特征。这些研究表明，模板碱基可以被移除，而不会失去剪切性-1/+1磷酸二酯键的切割特异性，而RNA引物中的对等损伤是抑制的。随后的研究确定了PPT引物的个别碱基，这些碱基对其功能至关重要。嘌呤类似物的使用也使我们能够确定这些关键位置上的嘌呤环的外环基团的作用。最后，使用HIV-1 RT的模拟取代底物和突变体的组合，核苷类似物被用来了解在访问HIV-1 RT的DNA聚合酶活性位点之前，单链模板如何与Finger亚域的残基相互作用。

Structural features of the polypurine tract primer of (+) strand DNA synthesis mediating its recognition by the RNase H domain of HIV-1RT have been examined. Such studies have indicated that template nucleobases can be removed without loss of cleavage specificity at the scissile -1/+1 phosphodiester bond, while equivalent lesions in the RNA primer were inhibitory. Subsequent studies identified individual bases of the PPT primer that are critical to its function. The use of purine analogs has also allowed us to determine the role of the exocyclic groups of the purine ring at these critical positions. Finally, using a combination of analog-substituted substrates and mutants of HIV-1 RT, nucleoside analogs have been used to understand how the single stranded template interacts with residues of the fingers subdomain prior to accessing the DNA polymerase active site of HIV-1 RT.