IN VITRO ANALYSIS OF A DNA REPLICATION APPARATUS

计划使用纯化的T4噬菌体复制蛋白

It is planned to use purified replication proteins of bacteriophage T4 to analyze the mechanisms involved in generation of high fidelity during DNA replication. The accuracy of T4 DNA polymerase (in the absence of assessory proteins) during the duplication of natural DNA templates will be measured in a number of different DNA sequence contexts. The influence of a variety of different factors, with special emphasis on polymerase accessory proteins, on the accuracy of this reaction will be measured. The role of these proteins will also be investigated by substituting mutant species in DNA replication reactions catalyzed by a seven enzyme replication complex. Attempts will be made to analyze the relative contributions of base mispairing, base stacking, efficiency of proofreading and longer range DNA sequence factors on the accuracy of DNA replication. An in vitro system for the study of frameshift mutations will be established so that the mechanisms involved in their generation can be analyzed in great detail.