Chapter Four DNA Replication

However, the "Central Dogma" has had to be revised a bit.  It turns out that you CAN go back from RNA to DNA, and that RNA can also make copies of itself.  It is still not possible to go from Proteins back to RNA or DNA, and no known mechanism has yet been demonstrated for proteins making copies of themselves.

(Reverse transcriptase in retroviruses)

DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle.

Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr

4.1 4.1 Three models for the replication of DNA

4.2 4.2 replication origin 、 direction & speed

4.3 Proteins of DNA Replication

4.4 DNA synthesis is semidiscontinuous

4.5 The precess of DNA replication of E.coli

4.6 circular DNA replication

4.7 linear DNA replication

4.8 The regulation of DNA replicaiton

4.14.1 Three models for the replication of DNA

①semiconservative model

②conservative model

③dispersive model

Three models for the replication of DNA

semiconservative replication:

conservative model:

dispersive model:

（ 1958 Meselson 和 Stahl ）：

CsCl gradient centrifuge

The Meselson-Stahl experiment, which showed that DNA replicates semiconservatively

P39

“ Heavy” DNA

“ Hybrid” DNA “ light” DNA “Hybrid” DNA

4.2 replication origin 、 direction & speed Replicon : The unit of DNA in which an individual act of replication occurs is called the replicon. (A unit of the genome in which DNA contain a region from origin to terminator ) Each replicon "fires" once and only once in each cell cycle.

The speed of replication

The speed of replication

Individual replicons in eukaryotic genomes are relatively small, typically ~40 kb in yeast or fly, ~100 kb in animals cells.

The rate of replication is ~2000 bp/min, which is much slower than the 50,000 bp/min of bacterial replication fork movement.

Electron Microscopy of replicating DNA reveals replicating bubbles.

From the speed of replication, it is evident that a mammalian genome could be replicated in ~1 hour if all replicons functioned simultaneously.

But S phase actually lasts for >6 hours in a typical somatic cell, which implies that no more than 15% of the replicons are likely to be active at any given moment.

theta

12

origin

2 1

O

origin

2 1

O

O

212 1

O

O

3’

3’Direction

DD

D

Prokaryotic cell Eukaryotic cell

Prokaryotic : a fixed origin Eukaryotic : a lot of origin

Eukaryotic replicons are 40-100 kb in length.

A chromosome is divided into many replicons.

Individual replicons are activated at characteristic times during S phase.

replication fork : The point at which replication is occurring is called the replication fork (sometimes also known as the growing point). A replication fork moves sequentially along the DNA, from its starting point at the origin. The origin may be used to start either unidirectional replication or bidirectional replication.

bidirectional replication

unidirectional replication

In unidirectional replication, one replication fork leaves the origin and proceeds along the DNA. In bidirectional replication, two replication forks are formed; they proceed away from the origin in opposite directions.

replication eye :

A molecule of DNA engaged in replication has two types of regions. the replicated region appears as a replication eye within the nonreplicated DNA. The nonreplicated region consists of the parental duplex; this opens into the replicated region where the two daughter duplexes have formed.

The appearance of a replication eye does not distinguish between unidirectional and bidirectional replication. As depicted in Figure, the eye can represent either of two structures. If generated by unidirectional replication, the eye represents one fixed origin and one moving replication fork. If generated by bidirectional replication, the eye represents a pair of replication forks. In either case, the progress of replication expands the eye until ultimately it encompasses the whole replicon.

A replication eye forms a theta structure in circular

DNA.

When a replicon is circular, the presence of an eye forms the θ-structure

4.3 Proteins of DNA Replication

DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication,a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed.

① DNA Helicases –

These proteins bind to the double stranded DNA and stimulate the separation of the two strands.

②DNA Topoisomerase –

This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process.

UnwoundParentalDuplex

Over-Woundregion

③DNA single-stranded binding proteins These proteins bind to the DNA as a

tetramer and stabilize the single-stranded structure that is generated by the action of the helicases.

Replication is 100 times faster when these proteins are attached to the single-stranded DNA.

single stranded DNA-binding protein, SSB

(1) SSB is consist of 177 amino acid residues in E.coli

(2) tetrapolymer

(3) a SSB= 32 nucleotides

(4) Cooperative binding

In addition to these proteins, several other enzymes are involved in

bacterial DNA replication.

④ DNA Polymerase of E.coli

DNA Polymerase –

DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase III (Pol III).

He and Severo Ochoa were the first to identify the enzyme catalyzing the synthesis of DNA, polymerase I. In recognition of their work elucidating the basic mechanisms of DNA replication, they were awarded the Nobel Prize in 1959.

Arthur Kornberg (1918 - )

a single polypeptide of 103 kD

The chain can be cleaved into two parts by proteolytic treatment.

The larger cleavage product (68 kD) is called the Klenow fragment .It contains the polymerase and the 3 –5 exonuclease activities.

The small fragment (35 kD) possesses a 5 –3 exonucleolytic activity, which excises small groups of nucleotides, up to ~10 bases at a time.

5' to 3' elongation

3' to 5' exonuclease

5' to 3' exonuclease

Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material.

Nick translation

DNA polymeraseIII  

900 kD

10 proteins

four types of subcomplex

Subunit

kDa Gene Subassembly

130 dnaE | 5'-3' polymerase 27.5 dnaQ

(mutD)| POL III CORE 5'-3'

exonuclease 10 | 3'-5'

exonuclease

71 dnaX

47.5 dnaX | 35 | ATP dependent' 33 | COMPLEX clamploader 15 | 12 |

40.6 dnaN CLAMP processivityfactor

DNA POLYMERASE III

four types of subcomplex There are two copies of the catalytic core. Each catalytic core contains the α subunit (the DNA polymerase activity), ε subunit (3 –5 proofreading exonuclease), and θ subunit (stimulates exonuclease). There are two copies of the dimerizing subunit, τ, which link the two catalytic cores together.

four types of subcomplex There are two copies of the clamp, which is responsible for holding catalytic cores on to their template strands. Each clamp consists of a homodimer of β subunits that binds around the DNA and ensures processivity. The γ complex is a group of 5 proteins, the Clamp loader, that places the clamp on DNA.

DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex that synthesizes the DNA of both new strands.

The holoenzyme assembles on DNA in three stages: First the clamp loader uses hydrolysis of ATP to bind β subunits to a template-primer complex.

The holoenzyme assembles on DNA in three stages: Binding to DNA changes the conformation of the site on β that binds to the clamp loader, and as a result it now has a high affinity for the core polymerase. This enables core polymerase to bind, and this is the means by which the core polymerase is brought to DNA.

The holoenzyme assembles on DNA in three stages: A τ dimer binds to the core polymerase, and provides a dimerization function that binds a second core polymerase (associated with another β clamp).

The b subunit of DNA polymerase III holoenzyme forms a ring completely surrounding a DNA duplex (shown in the center).

⑤ DNA Ligase – Nicks occur in the developing molecule because the

RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.

1) ATP+DNA ligase(E)→ E-AMP+PPi 。ATP is an integral part of the ligation reaction

2) E-AMP 上的 AMP 转移到 DNA 的 5′ 磷酸根上，使其活化，释放出酶。

3) 活化的 5′ 磷酸根与相邻的 3′ 羟基形成3′ ， 5′- 磷酸二酯键，并释放出 AMP 。

4.4 DNA synthesis is semidiscontinuous

The antiparallel structure of the two strands of duplex DNA poses a problem for replication. As the replication fork advances, daughter strands must be synthesized on both of the exposed parental single strands. The fork moves in the direction from 5 –3 on one strand, and in the direction from 3 –5 on the other strand. Yet nucleic acids are synthesized only from a 5 end toward a 3 end. The problem is solved by ?

leading strand:

On the leading strand DNA synthesis can proceed continuously in the 5 to 3 direction as the parental duplex is unwound.

lagging strand:

On the lagging strand a stretch of single-stranded parental DNA must be exposed, and then a segment is synthesized in the reverse direction (relative to fork movement). A series of these fragments are synthesized, each 5 –3 ; then they are joined together to create an intact lagging strand.

The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously.

复制机理的复杂性

Replisome

D.S. DNA

S.S. DNA 能量的供求多种酶类的互作复制的准确性（修复，校正）DNA聚合酶的一个共同特征是不能从头合成 DNA的一条链，合成新链的活动只能从一个已存在的 3‘端羟基开始，模板链必须转变成单链状态。

DNA synthesis occurs by adding nucleotides to the 3 -OH end of the growing chain, so that the new chain grows in the 5 →3 direction. The precursor for DNA synthesis is a nucleoside triphosphate, which loses the terminal two phosphate groups in the reaction.

A General Model for DNA Replication

1. The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase, DNA helicase and the single-stranded DNA binding proteins.

2. A free 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH of the primer is used to begin replication.

3. The replication fork moves in one direction, but DNA replication only goes in the 5' to 3' direction.This paradox is resolved by the use of Okazaki fragments. These are short, discontinuous replication products that are produced off the lagging strand. This is in comparison to the continuous strand that is made off the leading strand.

4. The final product does not have RNA stretches in it. These are removed by the 5' to 3' exonuclease action of Polymerase I.

5. The final product does not have any gaps in the DNA that result from the removal of the RNA primer. These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I.

6. DNA polymerase does not have the ability to form the final bond. This is done by the enzyme DNA ligase.

Synthesis of Okazaki fragments requires priming, extension, removal of RNA, gap filling, and nick ligation.

Model for the events occurring around a single replication fork of the

E. coli chromosome

Model for the events occurring around a single replication fork of the

E. coli chromosome (continued)

4.5 The precess of DNA replication of E.coli

OriC in E. coli chromosomal DNA

The four 9 bp consensus sequences on the right side of oriC

three A-T-rich 13 bp tandem repeats located in the left side of oriC.

Together the 9 bp and 13 bp repeats define the limits of the 245 bp minimal origin

•The position on the DNA at which replication start points are found.

* A DNA sequence that when added to a non-replicating DNA causes it to replicate.

* A DNA sequence whose mutation abolishes replication.

* A DNA sequence that in vitro is the binding target for enzyme complexes known to function in initiation of DNA replication.

Characteristics used to define Origins of Replication

isolation of ori

The precess of DNA replication of E.coli

① transcriptional activation

② Initiation of replication

Initiation of replication at oriC in vitro starts with formation of a complex that requires six proteins: DnaA, DnaB, DnaC, HU, Gyrase, and SSB.

Initiation complex formation DnaA protein binds to the 9 bp

consensus sequences cooperatively to form a central core around which oriC DNA is wrapped.

Then DnaA acts at three A-T-rich 13 bp tandem repeats.

In the presence of ATP, DnaA melts the DNA strands at each of these sites to form an open complex .All three 13 bp repeats must be opened for the reaction to proceed to the next stage.

④The prepriming complex

Altogether, 2-4 monomers of DnaA bind at the origin, and they recruit 2 complexes of DnaB-DnaC to bind, so that there is one for each of the two (bidirectional) replication forks.

Then DnaB uses its helicase activity to extend the region of unwinding.

Gyrase provides a swivel that allows one strand to rotate around the other; without this reaction, unwinding would generate torsional strain in the DNA. The protein SSB stabilizes the single-stranded DNA as it is formed.The protein HU is a general DNA-binding protein in E. coli . It stimulates the reaction.

⑤Primosome formationEach DnaB activates a DnaG primase, in one case to

initiate the leading strand, and in the other to initiate the first Okazaki fragment of the lagging strand.

⑥ DNA synthesis

Primase and Primosome

The primase is a RNA polymerase, which is different with that in the transcription. The primase synthesizes the primers, which is used in DNA synthesis The primer is a fragment of RNA The length of primer is 10-20bp approximately The helicases and other replicated factors have binded with DNA, then the primase have binds with them and formed a primosome at last.

回环模型 while one polymerase subunit synthesizes the leading strand continuously, the other cyclically initiates and terminates the Okazaki fragments of the lagging strand within a large single-stranded loop formed by its template strand.

Looping the lagging strand to make both polymerases move in the same direction

Termination in E. coli

The bacterial chromosome is replicated bidirectionally as a single unit from oriC. Two replication forks initiate at oriC and move around the genome (at approximately the same speed) to a meeting point.

replication fork trap

two termination regions

multiple terminators

Termination in E. coli

Sequences that cause termination are called ter sites.

A ter site contains a short (~23 bp) sequence

The termination sequences function in only one orientation.

The ter site is recognized by a protein (called Tus in E. coli and RTP in B. subtilis) that recognizes the consensus sequence and prevents the replication fork from proceeding

If for some reason one fork is delayed, so that the forks fail to meet at the usual central position, the more rapid fork will be trapped at the ter region to wait for the arrival of the slow fork.

4.6 circular DNA replication  rolling replication

D-loop

The rolling circle generates a multimeric single-stranded tail.

A rolling circle appears as a circular molecule with a linear tail by electron

microscopy.

φX174 RF DNA is a template for synthesizing single-stranded viral circles. The A protein nick the origin on the viral (+) strand and transfer to the new 5 end. At the same time, the released viral strand is circularized.

Rolling circles are used to replicate phage genomes

Replication circles for φX174 DNA

The displaced (+) strand may follow either of two fates after circularization. During the replication phase of viral infection, it may be used as a template to synthesize the complementary (–) strand. The duplex circle may then be used as a rolling circle to generate more progeny. During phage morphogenesis, the displaced (+) strand is packaged into the phage virion.

Mitochondria use different origin sequences to initiate replication of each DNA strand. Replication of the H-strand is initiated in a D-loop. Replication of the L-strand is initiated when its origin is exposed by the movement of the first replication fork.

D- 环型（ D-loop)

4.7  The ends of linear DNA are a problem for replication

We do not know whether initiation right at the end of a linear DNA is feasible. We usually think of a polymerase as binding at a site surrounding the position at which a base is to be incorporated.

3’-OH ?

3‘-OH

Lagging strand of circle DNA replication

Lagging strand of linear DNA replication

But

3‘

5‘

5‘

3‘

3’-OH ?

So a special mechanism must be employed for replication at the ends of linear replicons.

A protein may intervene to make initiation possible at the actual terminus. Several linear viral nucleic acids have proteins that are covalently linked to the 5 terminal base. The best characterized examples are adenovirus DNA, phage φ29 DNA, and poliovirus RNA.

In the case of adenovirus, a terminal protein is linked to the mature viral DNA via a phosphodiester bond to serine, as indicated in.

Terminal proteins enable initiation at the ends of viral DNAs

Adenovirus terminal protein binds to the 5 end of DNA and provides a C-OH end to prime synthesis of a new DNA strand.

The terminal protein has a dual role: it carries a cytidine nucleotide that provides the primer; and it is associated with DNA polymerase.

strand displacement mechanism The same events can occur independently at either end. Synthesis of a new strand starts at one end, displacing the homologous strand. When the replication fork reaches the other end of the molecule, the displaced strand is released as a free single strand. It is then replicated independently; this requires the formation of a duplex origin by base pairing between some short complementary sequences at the ends of the molecule.

4.8 the regulation of DNA replication

Replication starts with the transcription of an RNA that initiates 555 bp upstream of the origin. Transcription continues through the origin. The enzyme RNAase H cleaves the transcript at the origin. This generates a 3 –OH end that is used as the "primer" at which DNA synthesis is initiated

Two regulatory systems exert their effects on the RNA primer.

One involves synthesis of an RNA complementary to the primer;

the other involves a protein coded by a nearby locus.

The regulatory species RNA I is a molecule of ~108 bases, coded by the opposite strand from that specifying primer RNA. The relationship between the primer RNA and RNA I is illustrated in Figure . The RNA I molecule is initiated within the primer region and terminates close to the site where the primer RNA initiates. So RNA I is complementary to the 5 –terminal region of the primer RNA. Base pairing between the two RNAs controls the availability of the primer RNA to initiate a cycle of replication

The sequence of RNA I is complementary to the 5 region of primer RNA.

Base pairing with RNA I may change the secondary structure of the primer RNA sequence and thus prevent cleavage from generating a 3 -OH end.

Binding between RNA I and primer RNA can be influenced by the Rom protein, coded by a gene located downstream of the origin. Rom enhances binding between RNA I and primer RNA transcripts of >200 bases. The result is to inhibit formation of the primer.

Summary:

Replication of ColE1 requires transcription to pass through the origin, where the transcript is cleaved by RNAaseH to generate a primer end.

The regulator RNA I is a short antisense RNA that pairs with the transcript and prevents the cleavage that generates the priming end.

The Rom protein enhances pairing between RNA I and the transcript.

4.9 真核生物 DNA与原核生物 DNA复制的区别 1. 真核生物每条染色体上可以有多处复制起始点，而原核生物只有一个复制起始点。2. 真核生物的染色体在全部完成复制之前，各个起始点上 DNA的复制不能再开始，而在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，表现为只有一个复制单元，但可有多个复制叉。3. 原核生物只有一个复制子，真核生物可有多个复制子。

4. 真核生物 DNA复制所需酶及蛋白与原核生物的有所不同，真核生物 DNA聚合酶为α、 β、 γ 、 δ 、ε ， 低 等 真 核 生 物 有 三 种 DNA 聚 合 酶（ α 、 β 、 γ ），其中只有 δ 酶、 ε 具有 3 ’ →5 ’外切酶活性。而原核生物 DNA聚合酶为Ⅰ、Ⅱ、Ⅲ，且都具有 3 ’ →5 ’外切酶活性。真核生物 DNA 聚合酶 δ 和 ε 分别负责合成前导链和后随链，而原核生物 DNA聚合酶Ⅲ是两者兼顾。

5. 真核细胞内 DNA 引发酶和 DNA 聚合酶 α紧密偶联，而在原核细胞内引发酶和解链酶偶联在一起，形成复制体的一部分。 (DNA引发酶偶联对象不同）

练 习 题 一练 习 题 一1 、 DNA 复制时，下列哪一种酶是不需要的？ A DNA 指导下的 DNA 聚合酶 B DNA 连接酶 C 拓扑异构酶 D 解链酶 E 限制性内切酶

2 、合成 DNA 的原料是 A dNMA

B dNDP

C dNTP

D NTP

E NMP

3 、真核细胞进行 DNA 复制的部位是 A 细胞膜 B 细胞浆 C 细胞核 D 核蛋白体 E 微粒体 原核细胞进行 DNA 复制的部位是其拟核区

A 由 DNA 为模板合成的 DNA 片段 B 由 DNA 为模板合成的 RNA 片段 C 由 RNA 为模板合成的 DNA 片段 D 由 RNA 为模板合成的 RNA 片段

4 、 DNA 复制中的引物是

A 5’-TCTA-3’

B 5’-ATCT-3’

C 5’-UCUA-3’

D 5’-GCGA-3’

E 3’-TCTA-5’

5 、 DNA 复制时，模板序列 5’-TAGA-3’ 将合成哪种互补序列？

6 、 DNA 连接酶： A 使 DNA 形成超螺旋 B 使 DNA 链 上切口的两个末端连接起来 C 合成引物 D 将双螺旋解链 E 去除引物，填补空缺

7 、 Okazaki 片段是 A DNA 模板上的 DNA 片段B 引物酶催化合成的 RNA 片段C 随从链上合成的 DNA 片段D 领头链上合成的 DNA 片段

8 、 DNA 复制时，子链的合成方向是 A 一条子链 5’ 3’ ，另一条子链 3’ 5’

B 两条子链均为 5’ 3’

C 两条子链均为 3’ 5’

9 、 DNA 复制的特点是A 半保留复制B 需合成 RNA 引物C 形成复制叉D 有半不连续性E 形成 Okazaki 片段F 多点复制G 双向复制

14 、 DNA 复制时，连续合成的链为 ， 领头链而不连续合成的链为 。 随从链

dNTP

16 、 DNA 复制时，子链 DNA 合成的方向为 。 5’ 3’

17 、 DNA 复制时，亲代模板链与子代合成链之间碱 基配对的原则是： A 与 配对， C 与 配对。 T G

15 、 DNA 合成的原料是 。

18 、 DNA 复制时，改变模板 DNA 超螺旋结构的酶是 ，解开 DNA 双链的酶是 。 拓扑异构酶 解链酶

随从链19 、 DNA 的半不连续复制是指 的不连续合 成和 的连续合成。 领头链

20 、 DNA 的半保留复制是指复制所生成的子代DNA

分子中，其中有一条链是 ，而 另一条是 。

来自亲代 DNA新合成的

滚环复制（ rolling circle replication）又叫σ复制滚环复制有以下的特点：①共价延伸。θ复制是以引物的3‘-OH为起点以亲代链为模板从头合成一条新链，新链和旧链是分开的。但σ复制是先在一条亲代链（正链）上切一缺口，然后以另一 负环为模板，在正链切口上的 3′-OH上延伸，新合成的链和正链连在一道；

滚环复制有以下的特点：②模板链和新合成的链分开。 θ复制是半保留合成的，一条新合成的单链总是和模板链互补地结合在一起形成一条子链。而σ复制 3′端不断延环状模板合成， 5′端脱离负环，随着复制游离的 5′端越来越长，和环形模板完全分开；③不需 RNA引物，在正链 3′-OH上延伸；④只有一个复制叉，而θ复制一般都有两个复制叉。⑤形成多联体（ concatemer）

串联体模型：两个 5′有空缺的分子通过 3′端凸出的重复序列互补配对。然后 DNA多聚酶Ⅰ从 3′端延伸填补起这个空缺，留下最后的裂隙由连接酶封闭，产生了一个大分子串联体。再由限制酶在连接处交错切割，产生 3′ 凹端，由 DNA多聚酶Ⅰ在 3′-OH上延伸填满新的空缺，产生两条完整的双链。

??

concatermer

3‘-OH

ATCGTAGC

ATCGTAGC

3‘-OH