STRUCTURE AND REPLICATION OF DNA

这项建议中概述的研究的主要目标是 有助于理解DNA复制。我们的直接目标是 要确定在以下情况下发生的异常重新启动事件的原因 噬菌体伽马体在SOS条件下感染大肠杆菌。正常情况下，伽马 复制受到控制，以致于子代起源于复制 中间体处于非活动状态；只有在终止复制后，它们才会 支持启动下一轮复制。在SOS条件下， 然而，它们能够在一轮复制过程中启动； 显然，对启蒙的控制随后受到了深刻的干扰。 正常和异常启动事件的细节将在体内进行研究 而且是在体外。噬菌体和宿主内已知突变的影响将是 通过测定复制能力和分离研究(+SOS) 复制中间体。体外实验将涉及到质粒组 含有伽马起源的与启动子蛋白O(其 如果超螺旋张力在原点内产生展开的DNA区域 目前)以及与SOS反应相关的各种蛋白质。一个 HU和IHF蛋白在γ-和IHF中启动作用的比较 R6K也将被制造出来。这些研究将包括S1或P1核酸酶 灵敏度、EM和凝胶电泳法分析。这些研究将产生 关于更复杂系统中的DNA复制的信息。特别是，一个 了解SOS条件如何干扰启动的正常控制 复制对于最终理解正常和 高等生物体中的异常生长。 另一个主要目的是了解更多关于recA蛋白介导链的信息 交换反应。这项体外研究是与 考克斯博士将关注三链配对中间体和 也是一种新发现的链断裂反应，在某些情况下发生 链交换通过异质势垒时的条件。这 信息应该提供关于重组修复功能的见解 保护细胞免受DNA的诱变或致癌作用 损伤。 最后一个主要目标是关于FLP蛋白促进的位点特异性。 重组。将对反应中的中间体进行研究以获得 对Holliday结构异构化和DNA测序的洞察 调节反应。DNA-蛋白质、DNA-DNA和蛋白质-蛋白质 将对相互作用进行研究。这些信息最终应该是 与理解基因重组事件有关 监管。

The main objective of the studies outlined in this proposal is to contribute to the understanding of DNA replication. Our immediate aim is to determine the cause of the abnormal reinitiation events that occur when phage gamma infects E. coli under SOS conditions. Normally, gamma replication is controlled such that daughter origins within replicative intermediates are inactive; only after termination of replication do they support initiation of further rounds of replication. Under SOS conditions, however, they are able to initiate during a round of replication; apparently the control of initiation is then profoundly disturbed. Details of normal and abnormal initiation events will be studied in vivo and in vitro. Effects of known mutations within the phage and host will be studied (+ SOS) by determination of replication ability and isolation of replicative intermediates. In vitro experiments will involve plasmid containing the gamma origin reacted with the initiator protein O (which produces an unwound DNA area within the origin if superhelical tension is present) along with various proteins connected with the SOS response. A comparison of the effects of HU and IHF protein on initiation in gamma and R6K will also be made. The studies will include S1 or P1 nuclease sensitivity, EM and gel electrophoresis analysis. These studies will yield information on DNA replication in more complex systems. In particular, a knowledge of how SOS conditions disturb the normal control of initiation of replication is of importance for the eventual understanding of normal and abnormal growth in higher organisms. Another major aim is to learn more about the recA protein mediated strand exchange reaction. This in vitro study, carried out in collaboration with Dr. Cox, will be concerned with the triple-strand pairing intermediate and also a newly discovered strand breakage reaction that occurs under certain conditions when strand exchange passes a heterogeneous barrier. This information should offer insights on the recombinational repair function that guards cells against the mutagenic or carcinogenic effect of DNA lesions. The final major aim is concerned with FLP protein promoted site-specific recombination. The intermediates in the reaction will be studied to gain insights into the isomerization of Holliday structures and how DNA sequence modulates the reaction. DNA-protein, DNA-DNA and protein-protein interaction will be studied. This information should ultimately be relevant to an understanding of recombination events during gene regulation.