Are Membrane Proteins Directly Involved in Prokaryotic DNA Replication?

拟议的调查将严格分析与单链或双链克隆DNA插入物，来自B的复合物的几种膜蛋白的结合特性。subtilis复制起点一个65 kD的蛋白质，强烈结合两个相邻的起源插入，但不侧翼的起源区域或非起源DNA插入将被详细检查。将对蛋白质进行纯化，确认其膜形式，并确定参与蛋白质-DNA结合的特定序列。这种蛋白质与各种dna起始基因的关系将通过体外互补分析进行评估，使用从这些dna基因有条件缺陷的突变体中提取的DNA/膜亚复合物。dnaB基因将是特别感兴趣的研究，因为它已经被证明编码一种膜蛋白与复制起点DNA位点的亲和力。将用抗65 kD蛋白的抗体进行功能分析，以测量其阻断复制的能力。虽然膜相关DNA结合蛋白在复制过程中的功能目前还不确定，但它们在复制过程中的重要性已经确定。本提案中描述的新颖而严格的实验结果肯定会提供有关染色体复制过程中发生的事件序列的重要新信息。

The proposed investigations will critically analyze the binding characteristics of several membrane proteins that complex with single or ouble stranded cloned DNA inserts, derived from the B. subtilis origin of replication. One 65kD protein that binds strongly to two adjacent origin inserts, but not to flanking origin regions or non-origin DNA inserts will be examined in detail. The protein will be purified, its membrane form confirmed, and a determination will be made of specific sequences involved in the protein-DNA binding. The relationship of this protein to various dna initiation genes will be assessed by in vitro complementation analysis using DNA/membrane subcomplexes extracted from mutants conditionally defective in such dna genes. The dnaB gene will be of particular interest to study because it has already been shown to encode a membrane protein with affinity for replication origin DNA sites. Functional analyses will be done with antibodies against the 65kD protein, to measure their ability to block replication. Although the function of membrane associated DNA binding proteins in the process of replication is presently uncertain, their importance in the process is established. The results of the novel and rigorous experiments described in this proposal are certain to provide important new information on the sequence of events that occur during chromosome replication.