ENZYMOLOGY OF EUCARYOTIC DNA REPLICATION

拟议工作的目标是阐明这一机制(S) 在动物黑腹果蝇体内的DNA复制。这可能是 当纯化的酶在体外重组以产生 启动和维持特异性DNA合成的复制复合体 果蝇的DNA序列。 将采用生化和遗传相结合的方法来获得， 胚胎无细胞提取物的表征、分离和重组 能特异性复制重组DNA的黑腹果蝇 包含果蝇的染色体和线粒体DNA序列。这 将通过以下方式实现：(I)分子克隆和鉴定 果蝇复制的起源；(Ii)制备和 检测胚胎提取物启动和维持DNA的能力 复制；以及(Iii)与DNA合成有关的提纯酶 以研究它们的作用机制。 在正常发育期间对动物细胞繁殖的控制，以及 在癌症发展过程中失去控制，在 人类成长、衰老和疾病的过程。在每个单元格之前 分裂时，每个细胞的DNA互补被复制。因为DNA 复制和细胞分裂是紧密耦合的，理解 DNA在动物细胞中的复制机制对我们的 了解这些基本流程。

The objective of the proposed work is the elucidation of the mechanism(s) of DNA replication in the animal Drosophila melanogaster. This may be accomplished when purified enzymes are reconstituted in vitro to yield replication complexes which initiate and sustain DNA synthesis on specific Drosophila DNA sequences. A combined biochemical and genetic approach will be employed to obtain, characterize, fractionate, and reconstitute cell-free extracts of embryos of Drosophila melanogaster that specifically replicate recombinant DNAs containing Drosophila chromosomal and mitochondrial DNA sequences. This will be accomplished by: (i) molecular cloning and characterization of origins of replication from Drosophila melanogaster; (ii) preparing and examining embryo extracts for their ability to initiate and sustain DNA replication; and (iii) purifying enzymes implicated in the DNA synthesis reactions in order to study their mechanisms of action. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Prior to every cell division, the DNA complement of each cell is duplicated. Because DNA replication and cell division are tightly coupled, an understanding of the mechanism of DNA replication in animal cells is essential to our understanding of these basic processes.