分子生物学DNA的复制

All prokayotic chromosomes and many bacteriophage and
viral DNA molecules are circular and comprise(含) single
origin. Thus, there is a single termination site roughly 180o opposite the unique origin. Linear viral DNA usually have a single origin, but this is not necessarily in the centre of the molecules.



Speed
真核生物DNA的复制速度比原核生物慢，真核生物复制叉移动速度大 约为1000-3000 bp/min；细菌DNA 50000 bp/min，相差约20-50倍。 但真核生物染色体DNA上有许多复制起点，可以分段复制。真核生物 染色体DNA包含了许多串联排列的复制子，每一个复制子都有一个复 制起点，使真核生物染色体的复制实为多复制子的同步复制。
3.1.4 RNA priming
The leading strand and all lagging strand fragments are primed by synthesis of a short piece of RNA which is then elongated with DNA. The primers are removed and replaced
3.1.2 Replicons, origins and termini
3.1.3 Semi-discontinuous replication 3.1.4 RNA priming
3.1.1 Semi-conservative mechanism
During replication, the strands of the double helix separated
In contrast, the long, linear DNA of eukaryotic chromosomes
consist of multiple replicons, each with its own origin. Mammalian（哺乳动物） cell, 50000-100000 replicons
DnaB is a helicase. Helicases (解链酶) are enzymes which use the ennergy of ATP hydrolysis to move into and melt double-strand DNA (or RNA).
The single stranded bubble is coated with single-stranded binding protein (ssb) to protect it from breakage and to prevent the DNA renaturing. The enzyme DNA primase then attaches to the DNA and synthesizes a short RNA primer to initiate synthesis of the leading strand of the first replication fork.
The initiation of DNA replication always occurs at a fixed
point---origin. Usually, the replication forks proceed bidirectionally untill the teminus is reached.
Initiation at the E.coli origin involves wrapping of the DNA (four 9bp) around an initiator protein complex (DnaA-ATP) and separation of the strands at AT-rich sequences (three 13bp) which open to allow binding of DnaB protein.
② DNA解链酶（helicase）：DNA解链酶能通过水解ATP获得能 量来解开双链DNA，并在DNA上沿一定方向移动。大肠杆 菌的复制解链酶有DnaB、PriA和Rep蛋白，DnaB蛋白沿 5'→3'方向移动，PriA和Rep蛋白沿3'→5'方向移动。
3.2.2 Unwinding
For replication to proceed away the origin, DNA helicases must travel along the template strands to open the double helix for copying. Binding of Ssb protein further promotes unwinding. In a closed-circular DNA, removal of helical turn at the replication fork leads to the introduction of additional turns in the rest of the molecule in the form of positive supercoiling, which must be relaxed continuously by the introduction of further negative supercoiling by a type Ⅱ topoisomerase called DNA gyrase (旋转酶). Inhibitors of DNA gyrase are effective inhibitors of bacterial replication and have antibiotic activity. novobiocin(新生霉素) and
Bidirectional replication then follows.
补充一： ① DNA复制时，先由RNA聚合酶在DNA模板上合成一段RNA引物， 再由DNA聚合酶从RNA引物3'端开始合成新的DNA链。前导链 的合成与复制叉的移动同步。滞后链分段合成，需要不断 合成冈崎片段的RNA引物，然后由DNA聚合酶Ⅲ聚合DNA链。 ② 滞后链的引发往往由引发体（primosome）来完成。引发体 由DnaB、DnaC、DnaT、PriB、PriB、PriC六种蛋白质组成。 6种蛋白结合在一起形成引发前体，再与引发酶（DnaG）结 合，形成引发体，才能发挥功效。引发体沿复制叉运动方 向在DNA链上移动，并合成冈崎片段的RNA引物。DNA聚合酶 Ⅲ在模板链上合成冈崎片段，遇到上一个冈崎片段时则合 成停止。

复制方式
• 线性DNA双链的复制眼型 复制叉生长方式有单一起点的单向及双向 和多个起点的双向几种。DNA正在复制的部分在电镜下观察起来犹如 一只眼睛，称为复制眼。
• 环状DNA双链的复制：θ型，滚环型和D－环型
滚环复制 ΦΧ174 单向复制
D—环复制（D—loop） 存在于线粒体和叶绿体，其特点是两条 链复制不同步
补充二：
① 单链结合蛋白（single-strand binding protein, SSB）： SSB蛋白的作用是使被解开的单链在复制完成前能保持单 链结构，从而便于以之作为DNA合成的模板。SSB与单链 DNA的结合有高度协同性（cooperativity），即SSB的初步 结合能使其随后结合更为容易。SSB可以重复利用。
3．DNA replication
Including
3.1 An overview
3.2 Bacterial DNA replication
3.3 Eukaryotic DNA replication
3.1 An overview
3.1.1 Semi-conservative mechanism
3.1.3 Semi-discontinuous replication
Semi-discontinuous replication model. One strand, the
leading strand, is made continuously in a 5'→3' direction
from the origin. Synthesis of the second, lagging strand is then initiated at the replication fork and proceeds 5'→3' back towards the origin to form the Okazaki fragment. (1000-2000nt pro; 100-200nt eu) Soon after synthesis, the fragments are joined to make one continuous DNA by the enzyme DNA ligase（DNA连接酶）.
3.2.3 Elongation (延伸) 3.2.4 Termination （终止）
Experimental system
Genetically smaller and simpler simple bacteriophages (噬 菌体) and plasmids （质粒）are useful model systems for studying the in vitro replication of the large and fragile bacterial chromosome. Simple phageφx174 provides the best model for the replication of E. coli chromesome since it relies almost exclusively (主要依赖于) on host cellular replication factors for its own replication. Its replicative form comprises a supercoiled circle of only 5 kb.
3.2.1 Initiation
The E.coli origin, ori C, has been cloned into plasmids to produce more easily studied minichromosomes which behave like the E.coli chromosome.
This semi-conservative mechanism can be demonstrated
experimentally in 1958 by Meselson and Stahl.
3.1.2 Replicons, origins and termini
Any piece of DNA which replicates as a single unit is called a replicon.
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Direction
DNA复制从特定的位置开始，大多数是双向等速复制方向进行的。有 一些是单向的；或以不对称的双向方式进行。
Bidirectional replication of a circular bacterial repicon.(P76) Multiple eukaryotic replicons.
and each acts as a template to direct the synthesis of a new complementary daughter strand following the normal basepairing rules. Thus, each daughter cell receives one of the original parental strands. The point at which separation of the strands and synthesis of new DNA takes place is known as replication fork.
by DNA before ligation. This mechanism helps maintain high
replicational fidelity(保真性).
3.2 Bacterial DNA replication
3.2.1 Initiation (起始)
3.2.2 Unwinding（解旋）