NUCLEOTIDE BINDING PROPERTIES OF SV40 LARGE T PROTEIN

我打算检测SV40大T细胞的核苷酸结合特性 并确定它们与T功能的关系。SV40 T已知为 参与SV40 DNA复制的启动事件，但即使 虽然我们对T和SV40 DNA形成的复合体了解很多 起源我们不能解释启动的实际机制。确实有 据报道，T的许多修饰包括磷酸化，乙酰化， ADP-核糖化和寡聚，其中一些已被发现 影响T.的生化活性从我对 大T的结构-功能关系，我发现一个 核苷酰-蛋白质复合体在部分细胞孵育后形成。 纯化的SV40T和ATP在镁存在的情况下。三磷酸腺苷是 首选核苷酸，Alpha32P-ATP、Alphathio35S-ATP和~3H-ATP均为 标记为SV40的大型T。在本赠款提案中，概述了 确定T和核苷酸之间的化学键的性质， 并用于鉴定与该连接有关的氨基酸。装订 可以绘制蛋白质上的位点，并与目前的缺失进行比较 SV40大T相对于功能结构域的图谱。那我就会 寻求表明T可能作为核苷酸功能的初步数据 转移酶。在用放射性物质形成T-核苷酸络合物后 三磷酸腺苷，放射性标记可以从复合体中释放出来 镁、焦磷酸盐(PPI)和PolydT。没有重大的发布 在有镁存在的情况下用a)单磷酸，b)不含PPI的PolydT标记， 或c)ppi和PolydG、-dc或-da。这份拨款提案概述了方法 来证实这一观察结果。如果是这样，T和T之间的相互作用 宿主细胞复制酶可构成底物或底物酶 参与了第一个腺嘌呤核苷酸的启动 网站(S)。涉及核苷酸结合位点的定点突变 T和突变病毒的重组将允许分析 这种修饰在体内和生产中的功能意义 用于生化分析的突变体T。追踪推定的核苷酸 转移反应可能有助于解决T诱导的现象 从SV40T的转化功能出发启动DNA复制

I intend to examine the nucleotide binding properties of SV40 large T protein and determine their relationship to T function. SV40 T is known to be involved in the initiation events for SV40 DNA replication, but even though we know a great deal about the complex formed by T and the SV40 DNA origin we cannot explain the actual mechanism of initiation. There are many modifications reported for T including phosphorylation, acetylation, ADP-ribosylation, and oligomerization, some of which have been found to affect the biochemical activity of T. From my research into the structure-function relationships of large T, I discovered that a nucleotidyl-protein complex was formed following incubation of partially purified SV40 T and ATP in the presence of magnesium. ATP was the preferred nucleotide, and Alpha32P-ATP, Alphathio35S-ATP and 3H-ATP all labeled SV40 Large T. In this grant proposal methods are outlined for determining the nature of the chemical linkage between T and nucleotide, and for identifying the amino acid involved in the linkage. The binding site on the protein can be mapped and compared with the present deletion map of SV40 large T with respect to functional domains. Then I shall pursue preliminary data which suggest that T may function as a nucleotidyl transferase. After forming the T-nucleotidyl complex using radioactive ATP, radioactive label can be released from the complex in the presence of Mg, pyrophosphate(PPi) and polydT. There is no significant release of label in the presence of Mg with a) monophosphate, b) polydT without PPi, or c) PPi and polydG, -dC, or -dA. This grant proposal outlines methods of substantiating this observation. If so, the interaction between T and host cell replication enzymes could constitute that of a primer or primase involved in bringing the first adenine nucleotide to the initiation site(s). Site directed mutagenesis involving the nucleotide binding site of T and reconstitution of mutant virus would allow analysis of the functional significance of this modification in vivo as well as production of mutant T for biochemical analysis. Tracing the putative nucleotide transfer reaction might help to resolve the phenomenon of T-induced initiation of DNA replication from the transformation function of SV40 T.