北大课件3原核生物dna的复制1.ppt

原核生物DNA的复制 Replication of the E. coli chromosome In 1963, John Cairns’ Technique: Grew E. coli in 3H thymidine Waited till cells were in the middle of replication Lysed the cells very very gently Spread the lysate on an EM grid Exposed the grid to X-ray film for TWO months. What Cairns’ experiment (1963) showed: What Cairns did not show: DNA Synthesis 由于DNA双螺旋的两条链是反向平行的，因此两个模板极性不同。 所有已知DNA聚合酶的合成方向都是5’→3’， The short discontinuous segments are called Okazaki Fragments.? In bacteria they are approximately 1000 nt in length; in eukaryotes they are approximately 200 nt in length. DNA 复制的体系 亲代DNA分子为模板 四种脱氧三磷酸核苷(dNTP)为底物 提供3’-OH末端的引物 多种酶及蛋白质 DNA拓扑异构酶、DNA解链酶、单链结合蛋白、引物酶、 DNA聚合酶、RNA酶以及DNA连接酶等 DNA复制的基本过程 复制的起始 (initiation) DNA链的延伸 (elongation) 复制的终止 (termination) 1. DNA复制的起始 复制起始原点 DNA双螺旋的解旋 复制的引发 参与DNA复制起始和引发的蛋白质 DNA解旋酶(DNA helicase) 催化DNA双链的解链过程。 单链DNA结合蛋白(single strand DNA binding protein)： 以四聚体形式存在于复制叉处，只保持单链的存在，并不能起解链作用。 DNA拓扑异构酶(DNA topoisomerase) 消除DNA双链的超螺旋堆积。 引物酶(primase) 合成一小段RNA引物，为DNA新链的合成提供3’-OH末端。 DNA helicases separate the two strands of the double helix Binding of SSB to DNA inhibits the formation of intramolecular base pairs Action of topoisomerase at the replication fork DNA polymerase requires a 3’-OH end to initiate replication Substrates required for DNA synthesis DNA链的延伸需要的蛋白质: DNA聚合酶 滑动夹（Sliding DNA clamp） RNA酶(RNase H 等) 在复制完成后切除RNA引物。 DNA连接酶 (DNA ligase) 通过生成3’5’-磷酸二酯键连接两条DNA链。 DNA polymerase use a single active site to catalyze DNA synthesis Processivity (持续合成能力) Processivity is a characteristic of enzymes that operate on polymeric substrates. For DNA polymerase, the degree of processivity is defined as the average number of nucleotides added each time the enzyme binds a primer:template junction (a few~50,000). Structure of a sliding DNA clamp Sliding clamps dramatically increase DNA polymerase processivity (持续合成能力) The composition of the DNA Pol III holoenzyme The “trombone” model for coordina