Factors Influencing Genetic Transcription Initiation And

各种生物体都至少有一种降解RNA-DNA杂交体的RNA的酶。这种杂合体在体内由转录产生，并且通常与DNA复制相关，甚至在逆转录病毒（包括HIV）的复制中。到目前为止，这些核糖核酸酶H（RNases H）根据一级氨基酸序列相似性分为两类。从我们的研究中，我们知道，充分表征的大肠杆菌RNase HI在许多不同的物种，包括人类和小鼠中具有同源物。我们知道，这些哺乳动物蛋白质在序列和功能上类似于酿酒酵母的RNA酶H1，除了RNA酶H活性外，还具有双链RNA结合活性。类似地，细菌RNase HII蛋白在真核生物中具有对应物。RNA酶H执行重要的细胞功能，重要的是要知道这些酶是如何合成的。例如，在基于阿糖胞苷的反义疗法中使用DNA药物依赖于内源性RNA酶H来降解某些引起疾病的mRNA（在不适当的时间或位置合成的RNA）。提高靶细胞中RNase H活性的能力可以使这些反义DNA更有效的药物。类似地，某些类型的靶向抑制HIV逆转录酶的RNase H活性的药物也可能抑制细胞酶，导致不期望的效果。今年，我们在研究S.酿酒酵母和了解更多关于反义DNA寡核苷酸的细节。 我们以前报道过RNH 2L基因的转录随着细胞周期的进展而波动。我们注意到该基因上游的DNA元件在S.在整个基因组中出现110次，只有29次，在这种情况下，它可以积极调节相邻基因的转录。该元件包含两个重叠的DNA序列识别的转录因子负责在细胞周期的S-和G2/M期的表达，我们已经证明，两者都是用来帮助RNH 2L基因的转录。DNA元件序列的改变表现在其他基因的修饰表达模式以及RNH 2L基因本身的表达中。也许其他28个基因中的一些与这种重叠的启动子元件也受到类似于RNH 2L的调控。 从S.酿酒酵母具有高水平的RNase H活性，但在E.大肠杆菌无活性。我们发现其他多肽与活性形式的酶共纯化，表明多个亚基包含活性酶。RNH 2L基因表达的细胞周期调节以及由于重叠DNA位点引起的差异表达表明蛋白质在细胞周期的不同阶段可能具有不同的亚基以参与DNA复制或DNA修复。这些结果表明RNase H2 Lp的诱导或表达可能不足以增加与该多肽相关的RNase H活性。事实上，当RNA酶H2 Lp的过表达确实发生时，我们发现RNA酶H活性仅适度增加。 为了更好地理解RNA酶H如何识别RNA-DNA杂交体，从而帮助我们使用基于反义的疗法，我们对DNA组分进行了几种新的修饰。一种类型的修饰是在DNA寡核苷酸的3 '端添加各种取代基。观察到的RNA-DNA杂交体的RNA切割位点的差异告诉我们更多关于RNase H与RNA-DNA杂交体的相互作用。 今年的这些结果有助于我们了解这些重要蛋白质的作用机制

Various organisms have at least one enzyme that degrades the RNA of RNA-DNA hybrids. Such hybrids result in vivo from transcription and often are associated with DNA replication, even in replication of retroviruses including HIV. These ribonucleases H (RNases H), so far, fall into two classes based upon primary amino acid sequence similarity. From our studies, we know that the well-characterized Escherichia coli RNase HI has homologs in many different species including human and mouse. We know that these mammalian proteins resemble in sequence and function the RNase H1 of Saccharomyces cerevisiae by having a double-stranded RNA-binding activity in addition to the RNase H activity. Similarly, the bacterial RNase HII protein has counterparts in eukaryotes. RNases H perform important cellular functions and it is important to know how and by what means these enzymes are synthesized. For example, the use of DNA drugs employed in oligonucleotide-based antisense therapy relies on endogenous RNases H to degrade certain disease causing mRNAs (RNAs synthesized at inappropriate times or locations). The ability to increase RNase H activity in target cells could make these antisense DNAs more effective drugs. In a similar vein, certain types of drugs targeted to inhibit the RNase H activity of HIV reverse transcriptase could also inhibit the cellular enzymes leading to undesired effects. This year we have made considerable progress in examining the regulation and activity of RNase H2 of S. cerevisiae and also in learning more about the details of antisense DNA oligonucleotides. We previously reported that transcription of the RNH2L gene fluctuates in amounts as the cells progresses through the cell cycle. We noted that a DNA element upstream of the gene is relatively rare in S. cerevisiae occurring 110 times in the entire genome and only 29 times in a manner in which it can positively regulate transcription of the adjacent gene. This element contains two overlapping DNA sequences recognized by transcription factors responsible for expression in S- and G2/M phases of the cell cycle and we have demonstrated that both are used to aid transcription of the RNH2L gene. Alterations in the DNA element sequence manifest themselves in modified expression patterns of other genes as well as in the expression of the RNH2L gene itself. Perhaps some of the other 28 genes with this overlapping promoter elements are also regulated similar to RNH2L. The RNase H2Lp protein purified from S. cerevisiae has high levels of RNase H activity yet the same protein expressed in E. coli is inactive. We find other polypeptides co-purify with the active form of the enzyme suggesting that multiple subunits comprises the active enzyme. Cell-cycle regulation of the RNH2L gene expression together with the differential expression due to the overlapping DNA sites suggests the protein may have different subunits at different stages of the cell-cycle to participate in either DNA replication or DNA repair. These results indicate induction or expression of the RNase H2Lp may not be sufficient for increasing RNase H activity associated with this polypeptide. In fact, when overexpression of the RNase H2Lp does occur, we have found only a modest increase in RNase H activity. To better understand how RNA-DNA hybrids are recognized by RNases H, and thereby aid in our ability to use antisense-based therapies, we have made several novel modifications of the DNA component. One type modification was to add various substituents at the 3'-end of the DNA oligonucleotide. Differences in the site of cleavage of RNA of the RNA-DNA hybrid observed telling us more about the interaction of the RNase H with the RNA-DNA hybrid. These results from this year help us to understand the roles mechanism of action of these important proteins