STRUCTURE AND REPLICATION OF DNA

本建议中概述的研究的主要目标是 有助于理解DNA复制。 我们的近期目标是 为了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γ感染E.在SOS条件下， 一般来说，伽马 复制是受控的，这样子体起源于复制的 中间体是不活跃的;只有在复制终止后， 支持启动更多轮的复制。 在紧急呼救的情况下， 然而，它们能够在一轮复制期间启动; 显然，对启蒙的控制受到了严重的干扰。 将在体内研究正常和异常启动事件的详细信息 和体外培养。 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 复制中间体 体外实验将涉及质粒 含有与起始蛋白O（其 如果超螺旋张力被破坏， 沿着与SOS反应相关的各种蛋白质。 一 比较HU和IHF蛋白对γ和 R6 K也将被制造。 研究将包括S1或P1核酸酶 灵敏度、EM和凝胶电泳分析。 这些研究将产生 更复杂系统中的DNA复制信息。 特别是 SOS条件如何干扰正常控制的知识， 复制对于最终理解正常和 高等生物的不正常生长。 另一个主要目的是了解更多关于recA蛋白介导的链 交换反应 这项体外研究是与 博士考克斯，将关注三链配对中间体， 也是一种新发现的链断裂反应， 当链交换通过异质屏障时的条件。 这 这些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 病变 最后的主要目标是FLP蛋白的位点特异性启动 重组 反应中的中间体将被研究以获得 霍利迪结构的异构化以及DNA序列如何 调节反应。 DNA-蛋白质、DNA-DNA和蛋白质-蛋白质 将研究相互作用。 这些信息最终将 与理解基因重组过程中的重组事件有关， 调控

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.