The Replication of DNA

School of Life Science

Shandong University

Ch 8: The replication of DNA

1. Semiconservative replication

2. The chemistry of DNA synthesis

3. The mechanism of DNA polymerase

4. The replication fork

5. The specialization of DNA polymerases

6. DNA synthesis at the replication fork

7. Initiation of DNA replication

8. Finishing Replication

9. The regulation of genome replication

When Watson and Crick proposed the double stranded helix for the structure of DNA, they suggested that it might replicate by having the two parental strands separate and each one acting as a template for a new strand---- semiconservative replication.

1. Semiconservative replication

There are 3 possible mechanisms of DNA replication.

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Replication is semi-conservative

2. The chemistry of DNA synthesis

2.1 DNA synthesis requires deoxynucleoside triphosphates and a primer:template junction

A primer is a short piece of RNA that provides a 3’- OH for DNA synthesis.

p196 Fig.8-1

dNTP

2.2 DNA is synthesized by extending the 3’ end of the primer. Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis

p197 Fig. 8-2

5’ 3’

3. The mechanism of DNA polymerase

DNA polymerase resemble a hand that grips the primer:template junction

3.1 DNA Polymerase use a single active site to catalyze DNA synthesis.

Recognition of different dNTP by monitoring the ability of incoming dNTP in forming A-T and G-C base pairs (nearly identical geometry); incorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity.

p199 Fig. 8-3

Distinguishing between rNTP and dNTP by steric exclusion of rNTPs from the active site.

p199 Fig. 8-4

——通过动力学选择效应及空间排阻效应以保证参入碱基的正确性

细胞中rNTP的浓度比dNTP的高约10倍，

但前者的掺入速率却是dNTP的千分之一。

分辨器氨基酸

The palm domain binds two divalent metal ions (typically Mg+2 or Zn+2) that alter the chemical environment around the correctly base-paired dNTP and the 3’-OH of the primer

3.2 The palm domain of DNA Polymerase: Catalytic domain

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Each DNA polymerase has a characteristic processivity that can range from only a few nucleotides to more than 50,000 bases added per binding event.

Page206

3.3 DNA polymerases are processive enzymes

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3.4 Exonuclease proofread newly synthesized DNA

exonuclease: degrade from a end endonuclease: cut in the middle

Proofreading exonucleases work like a “delete key”

4. The replication fork

The junction between the newly separated template strands and the unreplicated DNA duplex is called the replication fork.

4.1 Both strands of DNA are synthesized together at the replication fork.

Leading strand

Lagging strand

The short fragments of new DNA in lagging strand are called Okazaki fragments and vary in length from 1000 to 2000 nucleotides in prokaryotes and from 100 to 400 nucleotides in eukaryotes .

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Fig. 8-11

3’

5’

5’

3’

Semidiscontinuous replication （半不连续复制）

——Okazaki fragments

Okazaki did his experiments in bacteriophage T4, which infects E. coli. He added [3H] thymidine to the infected E. coli cultures. Then the DNA was isolated and treated with a strong base to make it single stranded. It was separated by size using CsCl centrifugation. This allowed Okazaki to separate small pieces of DNA from larger ones.

The first few nucleotides at the 5’-end of Okazaki fragments are ribonucleotides. That is then removed before fragments are joined. Crucial for high fidelity of replication Primase (引发酶) is a specialized RNA polymerase making short RNA(5-10) primers that is complementary to one ssDNA. Remember that DNA polymerase requires a 3’ OH to begin DNA synthesis.

4.2 The initiation of a new strands DNA requires an RNA primer

The leading strand will need one primer and then the rest of the DNA template can be copied by DNA polymerase.

The lagging strand will require a primer for each Okazaki fragment.

4.3 RNA primers must be removed to complete DNA replication——RNase H

RNase H is a specific RNase that removes RNA from RNA:DNA hybrids. It leaves a the last RNA base pair (can only cleave bonds between two ribonucleotides), which must be removed by a 5’ exonuclease. (This 5’ exonuclease is part of DNA pol I in

E. coli.)

Figure 8-12

DNA pol I

4.4 DNA helicases unwind the double helix in advance of the replication fork

The DNA strands are separated by DNA helicase (解旋酶). It forms a ring around a single strand of DNA and slides along the DNA using energy from the hydrolysis of ATP. DNA helicase is also called DnaB (E. coli).

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4.5 Single-stranded binding proteins (SSBs) stabilize ssDNA prior to replication

SSBs bind to the single-stranded DNA to stabilize it. SSBs prevent the hydrogen bonds from reforming. The binding of a single SSB to the single-stranded DNA helps the binding of another SSB. This is called cooperative(协同作用) binding. Which helps the SSBs to quickly cover and stabilize all the single-stranded DNA. The SSBs do not bind to a specific sequence, but have sequence independent binding.—— Sequence-independent manner

Figure 8-15

4.6 Topoisomerase removes supercoils produced by DNA unwinding at the replication fork

As the strands of DNA are separated at the replication fork, the dsDNA in front of the fork develops positive supercoils.

Fig. 8-16

（复制机器）

Topoisomerase II

As DNA replication occurs positive supercoiling occurs. A type II topoisomerase (also called DNA gyrase) makes 2 cuts in the DNA to cause negative supercoiling to compensate for the positive supercoiling caused by replication.

Fig. 8-16

5. The specialization of DNA polymerases

There are different DNA polymerases that are specialized for different situations. Some are important for chromosome replication and others are important for DNA repair.

5.1 DNA polymerases are specialized for different roles in the cells

E. coli has 5 different kinds of DNA polymerases. They are named with Roman numbers in the order they were discovered. Each of the polymerases is specialized for a different role in the cell. Eukaryotic cells have multiple DNA polymerases. Three are essential to duplicate the genome: DNA Pol δ, DNA Pol ε and DNA Pol α / primase (引发酶)

DNA polymerase III (DNA Pol III) is the main enzyme that replicates the chromosomes. It is highly processive (once it binds to the DNA, it adds many base pairs before it dissociates).

DNA polymerase I (DNA pol I) is specialized for removing the RNA primers. It has a 5’ exonuclease that removes RNA or DNA directly in front of newly synthesized DNA.

The 5’ exonuclease can remove the RNA-DNA linkage that is

resistant to RNase H. (We will discuss the RNase H later.)

DNA pol I can also add nucleotides to the DNA strand. But it is not highly processive. It adds 20-100 nucleotides after binding. The short extent of synthesis is suited for replacing the region previously occupied by the RNA primers.

Both DNA pol I and DNA pol III are important for DNA replication. They must be very accurate, so both have a proofreading exonuclease.

The other 3 DNA polymerases in E. coli are important for DNA repair and lack proofreading capabilities.

Fig. 8-17 DNA polymerase switching during Eukaryotic DNA replication

DNA Pol α / primase (引发酶) primase: synthesize an RNA primer then replaced by DNA Pol δ (lagging strand), and DNA Pol ε (leading strand)

5.2 Sliding clamps (滑动加环/滑动钳) dramatically increase DNA polymerase processivity

Clamp protein slides along the DNA without dissociating from it.

Sliding DNA also bind tightly to DNA polymerases at replication forks.

After DNA polymerase has completed synthesis of the template, the absence of a primer:template junction causes a change in the DNA polymerase that releases it from the sliding clamp.

5.3 Sliding clamps are opened and placed on DNA by clamp loaders

ATP control of sliding DNA clamp loader

In E. coli, DNA Pol III holoenzyme（全酶） Enhance the function of core enzyme

Includes two copies of the “core” DNA Pol III enzyme and one copy of the five-protein γ complex

6. DNA synthesis at the replication fork

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Figure 8-22

E. coli trombone model

Protein interaction e.g. a) helicase—Pol III

holoenzyme b) helicase—primase

The combination of all the proteins that function at the replication fork is referred to as the replisome（复制体）, a finely tuned factory for DNA synthesis with the activity of each protein is highly coordinated.

Topoisomerase II

The initiation of replication requires the separation of the two strands of the DNA duplex to provide ssDNA.

DNA synthesis generally initiates at internal regions. The specific sites at which DNA unwinding and initiation of

replication occur are called origins of replication. All the DNA replicated from a particular origin of

replication is defined as a replicon.

7. Initiation of DNA replication

7.1 Specific genomic DNA sequences direct the initiation of DNA replication

Often there are other sequences besides the origin of replication that are important for initiating replication.

All the DNA sequences that are sufficient to direct the initiation of DNA replication are given the name replicator.

The initiator is a protein that specifically recognizes the DNA sequences in a replicator and initiates DNA synthesis.

7.2 The replicon model of replication initiation

Please read p230 Fig. 8-24

The E. coli replicator is called the OriC. It contains 5 sequences of 9 base pairs (called 9-mers) that are bound by the initiator DnaA.

Nearby there are 3 sequences called 13-mers that are A-T rich（easily unwound DNA）.

Fig. 8-25

7.3 Replicator sequences include initiator binding site and easily unwound DNA

Initiators have 3 functions: a) binding to specific

sequences in the replicator

b) Causing unwinding of the DNA helix

c) Binding to other proteins to bring them to the replicator.

oriC复制起点的解链过程

7.4 Protein-protein and protein-DNA interactions direct the initiation process

The initiator in eukaryotic cells is a 6 protein complex called the origin recognition complex (ORC). Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit. ORC recongnizes a conserved sequence A element, as well as a less- conserved B1 element. 整个细胞周期中均结合在ARS上. Binding of ORC does not cause the DNA strands to unwind. Binding of ORC does recruit other proteins that are necessary for replication to the replicator.

7.5 Eukaryotic chromosomes are replicated exactly once per cell cycle.

Replicators are inactivated by DNA replication----

Whether an origin is activated to cause its own replication or replicated by a replication for derived from an adjacent origin, it must be inactivated until the next round of cell division.

8.1 Type II DNA topoisomerases are required to Separate daughter DNA

Topoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

8. Finishing Replication

线性双链DNA分子连续复制可能变短的两个原因：

① 滞后链的最后片段不足以形成冈畸片段

② 即使足以形成冈畸片段其引物RNA的切除也能使DNA链变短

Fig. 8-35

8.2 Lagging-strand synthesis is unable to copy the extreme ends of linear chromosomes

端粒酶延伸G丰富链

Telomerase is a reverse transcriptase together with a template RNA

Telomerase is different from other DNA polymerases in that it does not require a DNA template.端粒酶由蛋白和RNA组成

Fig. 8-37

8.3 Telomerase is a novel DNA polymerase that does not require an exogenous template

The telomeres only acts on the 3’ end of the telomere. The 5’ end is accomplished by the lagging-strand DNA replication machinery.

Fig. 8-38

8.4 Telomerase solves the end replication problem by extending the 3’ end of the chromosome

9. The regulation of genome replication

复制起始后，或是DNA被修饰（甲基化和去甲

基化），或是特异性蛋白因子被修饰（磷酸化和去磷酸化等），这些修饰使得特异性蛋白因子对复制起始点的识别状态发生改变。

9.1 E. coli DNA的复制调控：

In E.coli , Dam methylase add methyl group to the A within every GATC.

The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis. ——半甲基化

The SeqAbind thoses methylated.——reduces the methylated rate and prevents DnaA from associating with oriC and initiating a new round of replication.

Reduce the initiation replication from new copies of oriC

Box 8-7, Fig. 1

原核生物的DNA链延伸的速度几乎是恒定的（ E.coli DNA完成复制的时间大约为40分钟），但细胞生长和增

殖速度取决于培养条件，营养丰富生长快速。

The mechanisms for prevent rapid

reinitiation do not last until cel division.

Box 8-7, Fig. 1

Please read the Box 8-7

Re-initiation of bacterial replication at new origins before completion of the first round of replication

生长、增殖速度不同的细胞中，

复制叉的数量不同，快速生长的原核生物中，复制起始点上可以连续开始新的DNA复制，表现为，虽然

只有一个复制子，但有多个复制叉，即一个复制循环没有完成，下一个复制循环就已经开始。

原核生物DNA的复制与细胞分裂一

般是同步的，但复制与细胞分裂不直接偶联，复制起始不依赖细胞分裂，而复制终止则能引发细胞分裂。

G1

M G2

S

9.2 真核细胞DNA的复制调控

——Eukaryotic chromosome are replicated exactly once per cell cycle

真核生物具有多个起始复制子，每一次DNA复制并不是所有的复制起始

点都需要启动。例如酿酒酵母基因组约有500个复制子，但每次DNA复制时只需要启动10－20％的复制起点，并且每次DNA复制启动的复制起始点是随机的。

Prereplicative complex formation is the first step in the initiation of replication in Eukaryotes

Fig. 8-30

The steps for Pre-RC formation:

① The ORC recognize and bind to the replicator.

② Two (Cdc6 and Cdt1) helicase loading proteins(解旋酶装卸蛋白)are recruited.

③ Then the eukaryotic replication fork helicase (真核复制叉解旋酶)Mcm2-7 complex is recruited.

④ Pre-RC formed in G1 phase

pre-replicative complex (Pre-RC)

Pre-RC are activated to initiate replication by two protein kinases ——Cdk(cyclin dependent kinase 细胞周期依赖性激酶) ——Ddk(Dbf4- dependent kinase )

Each of these kinases is activated only when the cell enter S phase Then the polymerases are recruited

Pre-RC formation and activation are regulated to allow only a single round of replication during each cell cycle

Fig. 8-33