分子生物学 ch 4 DNA replication.pptVIP

In E.coli，there is only one oriC （1） 含有多个短重复序列， （2） A-T rich, （3） 能被专一性的起始蛋白识别，形成replisome(复制体). Replication origin 复制起始点 Bidirectional Semi-discontinuous θ复制 环状DNA复制时状如θ，称θ复制，又叫Cairns复制。复制中间体称为Cairns分子。 primosome In molecular biology, a primosome is a protein complex responsible for creating RNA primers on single stranded DNA during DNA replication. The primosome consists of seven proteins: DnaG primase, DnaB helicase, DnaC helicase assistant, DnaT, PriA, Pri B, and PriC. The primosome is utilized once on the leading strand of DNA and repeatedly, initiating each Okazaki fragment, on the lagging DNA strand. (2) Elogation Replisome: the complex of key replication proteins with DNA at the replication fork Recent papers shed insight into the architecture and dynamics of the components of the bacterial replisome. Helicase Gyrase Primase DNA polymerase III holoenzyme DNA polymerase I Ligase (3) Termination 复制终止的序列ter与TUS蛋白结合 initiation 共价延伸 ? 主要指滚环复制（也称σ复制）；先导链基于一亲链基础上共价延伸。 Example 2 : ΦX174 Chapter 4 DNA replication I. Semi-conservative and semi-discontinuous 1. Semi-conservative replication (半保留复制) 　 1958年Meselson-Stahl实验 先将大肠杆菌放在15NH4C1培养基中生长15代，使几乎所有的DNA都被15N标记后，再将细菌移到只含有14NH4C1的培养基中培养。随后，在不同的时间取出样品，用十二烷基硫酸钠(SDS)裂解细胞后，将裂解液放在CsC1溶液中进行密度梯度离心(140000g，20小时)。离心结束后，从管底到管口，溶液密度分布从高到低形成密度梯度。DNA分子就停留在与其相当的CsC1密度处， 在紫外光下可以看到形成的区带。 The Meselson-Stahl Experiment Autoradiograph ，放射自显影 2. Semi-discontinuous replication 半不连续复制 前导链(leading strand) 滞后链(lagging strand) 冈崎片段(Okazaki fragment) DNA polymerase DNA 聚合酶 Template + primer + 4dNTP DNA polymerase dsDNA Direction: 5’ 3’ The direction of synthesis of the new DNA chain is always from 5’ to 3’. 5’ to 3’ Addition of Monomers At the growing end of the DNA chain, phosphodiester bond formation between the 3’-OH of the last nucleotide and the 5’-phosphate of the incoming dNTP . To synthesize a chain of DNA, DNA polyme