ENZYMOLOGY OF EUCARYOTIC DNA REPLICATION

拟议工作的目标是阐明机制 黑腹果蝇的DNA复制。 这可能是 当纯化的酶在体外重构以产生 启动和维持DNA合成的复制复合物， 果蝇DNA序列。 将采用生物化学和遗传学相结合的方法来获得， 表征、破碎和重构胚胎的无细胞提取物 特异性复制重组DNA 含有果蝇染色体和线粒体DNA序列。 这 将通过以下方式完成：（i）分子克隆和表征 来自黑腹果蝇（Drosophila melanogaster）的复制起点;（ii）制备和 检查胚胎提取物启动和维持DNA的能力 复制;和（iii）纯化涉及DNA合成的酶 反应，以研究其作用机制。 在正常发育过程中对动物细胞繁殖的控制， 在癌症发展过程中失去控制，是至关重要的， 人类的成长、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互补物被复制。 因为dna 复制和细胞分裂是紧密相连的，对细胞分裂的理解， 动物细胞中的DNA复制机制对我们的研究至关重要。 了解这些基本过程。

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.