Factors Influencing Genetic Transcription Initiation And

各种生物体至少有一种酶可以降解RNA- dna杂交体的RNA。这种杂交体在体内由转录产生，通常与DNA复制有关，甚至在包括艾滋病毒在内的逆转录病毒的复制中也是如此。到目前为止，这些核糖核酸酶H （RNases H）根据初级氨基酸序列的相似性分为两类。从我们的研究中，我们知道具有良好特征的大肠杆菌RNase HI在包括人类和小鼠在内的许多不同物种中具有同源物。我们知道这些哺乳动物蛋白在序列和功能上与酿酒酵母的RNase H1相似，除了具有RNase H活性外，还具有双链rna结合活性。类似地，细菌RNase HII蛋白在真核生物中也有对应物。rna酶H发挥着重要的细胞功能，了解这些酶是如何合成的以及通过什么方式合成的是很重要的。例如，基于寡核苷酸的反义治疗中使用的DNA药物依赖于内源性rna酶H来降解某些致病mrna（在不适当的时间或位置合成的rna）。在靶细胞中增加RNase H活性的能力可以使这些反义dna成为更有效的药物。类似地，某些类型的靶向抑制HIV逆转录酶RNase H活性的药物也可能抑制细胞酶，导致不希望的效果。

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. The RNase H2Ap 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 RNH2A 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 H2Ap may not be sufficient for increasing RNase H activity associated with this polypeptide. In fact, when overexpression of the RNase H2Ap does occur, we have found only a modest increase in RNase H activity. We have also found that the RNase H1 protein of human and mouse form a dimer in the presence of substrate and that this complex leads to processivity of the enzyme (i.e., the protein tends to degrade one molecule of substrate regradless of size before releasing and attacking a second molecule). These results from this year help us to understand the roles mechanism of action of these important proteins