Palaniappan C, Fuentes G M, Rodríguez-Rodríguez L, Fay P J, Bambara R A
Department of Biochemistry, University of Rochester, New York 14642, USA.
J Biol Chem. 1996 Jan 26;271(4):2063-70.
RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.
人类免疫缺陷病毒（HIV）复制过程中的RNA/DNA杂交体在由杂交体结构决定的位置被HIV-1逆转录酶（RT）H切割。负链DNA合成伴随着位于生长中的3' DNA末端的RT对模板病毒RNA的切割。一些RNA以寡聚体形式退火到新的DNA链上，并被位于5' RNA末端的RT切割。我们构建了底物来检验以下假设：内部螺旋结构而非链末端结构驱动RT定位在3' DNA和5' RNA末端。在DNA模板上有RNA引物凹陷的底物上，RNA的5'末端在RNase H切割位置的确定中起主导作用。如果RNA的5'末端区域不能退火，切割就不会发生。然而，我们获得的证据表明，螺旋结构促进RT与最靠近5' RNA/3' DNA末端的螺旋区域末端结合。当RNA模板上凹陷的DNA引物有一个3'未退火区域时，会发生切割，RT仅由杂交体退火区域5' RNA/3' DNA末端的螺旋结构定位。使用RNA引物退火到环状DNA模板的底物，我们表明切割可以独立于DNA 3'末端的存在，并由5' RNA末端引导。总体而言，结果表明RT最初结合杂交体的内部区域，然后朝着遇到3' DNA或5' RNA末端的方向移动，在那里它被链末端定位以进行催化。讨论了病毒复制中RNA切割的两种模式的要求以及对5' RNA末端结构的意外要求。
