DNA Replication, DNA Damage and Repair. Outline Central Dogma( 中心法则 ) DNA Replication( 复制 ) Testing Models for DNA replication Semi-conservative replication.

DNA Replication , DNA Damage and Repair

Outline• Central Dogma( 中心法则 )• DNA Replication( 复制 )• Testing Models for DNA replication• Semi-conservative replication( 半保留复制 )• Evidence for Semi-Discontinuous( 半不连续 ) Replication• E. coli DNA polymerases• Details of DNA Replication 　 Three steps• 1) Initiation （起始）• 2) Elongation （延伸）• 3) Termination and Separation• （终止与分离）• Eukaryotic DNA Replication Like E. coli, but more complex • DNA Damage and Repair• Types of Repair

Central　　　　Dogma

中心法则

Central Dogma

DNA→RNA→Protein

Central Dogma

Central Dogma

Transcription Translation

Replication Replication

Retro-transcription

Gene expressionCentral Dogma

转录（ transcription ） :

Protein synthesis is referred to as translation.

基因表达（ gene expression):Gene expression is the process by which a gene's information is converted into the structures and functions of a cell.

The process in which DNA is used to synthesize RNA is called transcription.

翻译（ translation):

Central Dogma

转录（ transcription ） :

序 论

是以 mRNA 为模板 , 按照三个核苷酸决定一个氨基酸的原则 , 把 mRNA 上的遗传信息转换成蛋白质分子中特定的氨基酸序列的过程。

基因表达（ gene expression):

DNA 把基因信息传递给 RNA 的过程。

翻译（ translation):

通过转录和翻译，用基因的遗传信息在细胞合成了有功能意义的各种蛋白质。

Coding strand, Sense strand, Crick strand

Template strand, antisense strand, Watson strand

Transcription

Translation

Central Dogma

Happy Birthday,Double Helix

DNA Replication

DNA Replication

In 1953, James Watson and Francis Crick deduced the SecondaryStructure of DNADouble-helix Structural Model ( 双螺旋结构模型 ) . This was one of the most important biological advances, since it led to an understanding of the relationship of the DNA structure to its function, particularly to the way it was replicated ( 复制 ).

DNA Replication

"Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid"

(Nature, April 25, 1953. volume 171:737-738.)

"...It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. The structure itself suggested that each strand could separate and act as a template for a new strand, therefore doubling the amount of DNA, yet keeping the genetic information, in the form of the original sequence, intact. "

DNA Replication

• Can you remember The Basic Features of B-DNA?

DNA Replication

• Can you remember The Basic Features of B-DNA?

• There have 5 features and 6 parameters.

DNA Replication

4. Bases are Restricted Pairing on the Inside

A purine-pyrimidine pair fits PERFECTLY. Three hydrogen bonds （氢键） hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the other pair, the A-T pair, is held together by two hydrogen bonds.

The Basic Features of B-DNA

5. A Pair of Chains are Complementary Sequences

The bases are complementary, with A on one side of the molecule you only get T on the other side, similarly with G and C. If we know the base sequence of

one strand we know its complement.

The Basic Features of B-DNA

DNA Replication　General　Features

DNA Replication

• DNA Replication General Features 1) Many enzymes and proteins are required

2) Template & dNTPs/Mg 2+ are required

3) Semi-conservative ( 半保留） A key experiment designed by M. Meselson and W. F.

Stahl （ 1958 ）

4) DNA Unwinding is necessary

5) A Primer ( 引物） with a free 3' -OH group is required

DNA Replication

DNA Replication General Features6) Only in the 5′→3′direction

7) Specific Origin of Replication-Ori C( 大肠杆菌的复制原点 )

8) Bi-directional (With some exceptions)

9) Semi-discontinuous ( 半不连续 ) Replication fork ( 复制叉 ) ， Leading strand

( 前导链 ), Lagging strand ( 后随链 ) and Okazaki fragments ( 冈崎片段 )

10) Highly processive ( 进行性 ), Highly ordered and Extremely accurate

DNA Replication

Testing Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)

DNA Replication

Matthew Meselson and Franklin Stahl more recently

Faculty member at HarvardMechanisms of Molecular Evolution

Faculty Chair for CBW Studies

Faculty member at U. of OregonMeiotic Recombination

DNA Replication

1958 年 Meselson 和 Stahl 用同位素示踪和密度梯度离心的方法证明了 DNA 的半保留复制

DNA Replication

Testing Models for DNA replicationMeselson and Stahl (1958)

Density labeling experiment on E. coli (bacterial) DNA

Bacterial culture

15NH4Cl(Sole N source)

Grow for several generations

Bacterial culturewith dense DNA

This is the starting material for the experiment

DNA Replication

Meselson and Stahl (continued)

Harvest cells and resuspend in media with14NH4Cl as the sole N source

Bacterial culturewith dense DNA

Grow for 1 generation

Harvest some cells“1st generation”

Grow for another generation

Harvest some cells“2nd generation”

Grow for another generation

etc

For each generation isolate the DNA and spin through a density (CsCl) gradient).Detect DNA in the gradient (by UV absorption)

Monitor how many DNA bands there are after each generation

Bacterial culture“0 generation”

14NH4Cl

DNA Replication

Meselson and Stahl Original DataDNA Replication

DNA Replication

Semi-conservative　replication?半保留复制的定义

Semi-conservative replication: After the two strands separate, each serves as a template for the synthesis of a complementary strand.(In other words, each of the two new DNA molecules contains one old strand and one new strand.) This process, referred to as semiconservative replication.

DNA Replication

• Since DNA replication is semiconservative, therefore the helix must be unwound.

DNA Replication

• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.

DNA Replication

John Cairns

Grow cells for several generationsSmall amounts of 3H thymidineare incorporated into new DNA

Grow for brief period of time

Add a high concentration

of 3H- thymidine

in media with lowconcentration of

3H- thymidine

Bacterial culture

*T

*T

*T

*T

Dense label at the replication forkwhere new DNA is being made

*T*T *T *T

*T*T

*T*T

*T*T*T

*T*T

*T*T*T

*T*T *T *T

*T*T*T*T

*T*T*T

All DNA is lightlylabeled with radioactivity

*T*T *T

Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

DNA Replication

Evidence points to bidirectional replication

Label at both replication forks

DNA Replication

DNA Replication

DNA Replication

DNA Replication is Semi-discontinuous

Consider one replication fork:

5’

3’

5’

3’

Direction ofunwinding

Continuous replication

5’

3’Primer

Primer

5’

3’

Primer

5’

3’Discontinuous replication

DNA Replication

在复制的起始点处， DNA 双链部分解开为单链，形成叉子形状称复制叉（ replication fork ）。

DNA Replication

Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)

DNA Replication

Evidence for Semi-Discontinuous Replication(pulse-chase experiment)

Bacteria arereplicating

Bacterial culture

Add 3H Thymidine

For a SHORT time(i.e. seconds)

Flood with non-radioactive T

Allow replicationTo continue

Harvest the bacteriaat different timesafter the chase

Isolate their DNASeparate the strands(using alkali conditions)Run on a sizing gradient

smallest

largest

Radioactivity will onlybe in the DNA that was made during the pulse

DNA Replication

smallest

largest

Results of pulse-chase experiment

Pulse

5’

3’

5’

3’

Direction ofunwinding

3’

5’

Primer

Primer

5’

3’

Primer

5’

3’

* * *

***

Chase

DNA Replication

Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous

DNA Replication

Many enzymes and proteins are required in DNA Replication.

DNA Replication

Many enzymes and proteins are required in DNA Replication.

Do you know how many enzymes are required in DNA Replication?

DNA Replication

Topoisomerase （拓扑异构酶） DNA　Helicase （ DNA 解链酶） Single-stranded　DNA　binding　proteins （ SSB ，单链结合蛋白）

Primase （引发酶） -　formation of RNA primers

DNA　–dependent　DNA　polymerase　III　(DNA　pol,　DNA 聚合酶 III ）

The　Enzymes　responsible　for　removing　RNA　primers　(DNA　polymerase　II))

 　DNA　ligase　 （ DNA　 连接酶） -joining of Okazaki fragments

Enzymes and Proteins Involved in DNA Replication

DNA Replication

DNA Replication

DNA Replication

E. coli DNA polymerases Identification

Kornberg and DNA pol I (Kornberg enzyme) Structure and Function of DNA pol I

A multi-functional enzyme DNA pol III is a major polymerase involved

in E. coli chromosome DNA replication

DNA Replication

DNA polymerase I （ pol I ）： 1956 年

Kornberg 等在 Ecoli 中发现的第一个 DNA

聚合酶，是 DNA 复制研究中的重要里程碑，因此于 1959 年 Kornberg 获得诺贝尔化学奖。

Currently a faculty member at Stanford School of Medicine

DNA Replication

DNA Pol I DNA Pol I from E. coli is 928 aa (109 kD) from E. coli is 928 aa (109 kD) monomer monomer A single polypeptide with at least A single polypeptide with at least three different Enzymatic activities!three different Enzymatic activities!

How Amazing!!!

a 3’ to 5’ exonuclease activity a 5’ to 3’ exonuclease activity a 5’ to 3’ DNA polymerizing activity

DNA Replication

Proof reading activityof the 3’ to 5’ exonuclease.

Proof reading activity is slowcompared to polymerizingactivity, but the stalling ofDNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizingactivity and remove theincorrect base.

DNA Replication

DNA Replication

DNA Replication

A total of 5 different DNAPs have been reported in E. coli

DNAP I: does 90% of polymerizing activity DNAP II: functions in DNA repair (proven in 1999)

DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in

1999)

DNAP V: functions in DNA repair (discovered in 1999)

The DNA Polymerase Family

DNA Replication

The "real" replicative polymerase in E. coli It’s accurate: makes 1 error in 107 dNTPs

added, with proofreading, this gives a final error rate of 1 in 1010 overall.

fast: up to 1,000 dNTPs added/sec/enzyme It’s highly processive: >500,000 dNTPs

added before dissociating

DNA Polymerase III

IT’S COMPLICATED!!!

DNA Replication

DNA Replication

The subunits of E. coli DNA polymerase III

Subunit Function

’

5’ to 3’ polymerizing activity3’ to 5’ exonuclease activity and assembly (scaffold)Assembly of holoenzyme on DNASliding clamp = processivity factorClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complex

CoreEnzymedimer

Ho

loen

zym

e

DNA Replication

• The structure formed by two beta subunits of  the E. coli DNA polymerase III .  This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule.

DNA Replication

DNA Replication

DNA Polymerase IIIholoenzyme

Core

Core

clamps

ReplicationFork

Leading Strand synthesis

Lagging Strand synthesis

DNA Replication

大肠杆菌三种 DNA 聚合酶的比较

pol I pol II pol III

分子量 103Kd 88Kd 900Kd

不同种类亚基数目 1 ≥ 7 ≥ 10

每个细胞的分子数 400 100 10

生物学活性 1 0.05 15

5'→3' 聚合酶活性 ＋ ＋ ＋

3'→5' 外切酶活性 ＋ ＋ ＋

5'→3' 外切酶活性 ＋ － －

功能 校正，修复，去除 RNA 引

物修复

复制（ 5'→

3' 聚合作用）

DNA Replication

DNA　Polymerase　III　is　principal　DNA　replication　enzyme　in　DNA　replication.

But　the　other　important　Enzymes　and　Proteins　Involved　in　DNA　Replication?

DNA Replication

Action of DNA LigaseDNA Replication

DNA Replication

DNA Replication

以 NAD+ 供能（细菌） :E+NAD+→E － AMP+NMN （烟酰单核苷酸）

连接切口（ DNA连接酶）

以 ATP供能（病毒，真核） : E+ATP→E － AMP+ppi

DNA　Replication　is　an　Ordered　Series　of　Steps.

The　Following　is　Details　of　DNA　Replication.

DNA Replication

Details of DNA Replication Three steps 1) Initiation （起始） 2) Elongation （延伸） 3) Termination and Separation （终止与分离）

DNA Replication

DNA Replication is an Ordered Series of Steps1) Initiation （起始）Find the originUnwind the helix2) Elongation （延伸）Synthesize primers ElongateRemove the primers and ligate Okazaki fragments3) Termination and Separation （终止与分离）Terminate replication Separate Daughter DNAs

DNA Replication

Find the origin: DnaA (origin recognition protein) + HU

DNA Replication-- Initiation

DNA Replication-- Initiation

Unwind the helix: DnaB (helicase), DnaC + DnaT (deliver DnaB to the origin), SSB (keeps helix unwound), DNA Gyrase facilitates efficient unwinding

DNA Replication-- Initiation

Finding and unwinding the origin of replication

13 base pair repeat =

5’-GATCNTNTTNTT-3’

4 DnaA tetramersfirst bind to the repeats.Binding is cooperative.Each DnaA binds ATP.

They recruit additional DnaA monomers to bind to adjacent DNA generating a nucleosome-like structure

DnaA powers the unwindingof adjacent A-T-rich repeatsby hydrolyzing ATP. A proteincalled HU also helps.

DNA Replication-- Initiation

DnaB ( a helicase), is now delivered tothe unwound region with the help ofDnaC and DnaT. You need one helicaseat each replication fork to do theunwinding. Delivery and assembly ofDnaB onto DNA requires ATP.

SSB coats the unwound DNA strandsto prevent them from reassociating.

Unwinding starts in both directions, andshoves off (displaces) the DnaA proteins.

This a prepriming complex.

DNA Replication-- Initiation

• Synthesize primers: DnaG (primase) + PriA, PriB,PriC (assembly and function of the primosome)

DNA Replication-- Elongation

Primase is now recruited to each forkso that a primer can be laid down for DNAsynthesis on each strand at each fork.

Primase is associated with helicase.Primase lays down an RNA primerRNA primer on the leading strand.

Primase lays down a primer on the laggingstrand.

This a primosome引物体

DNA Replication-- Elongation

Elongate (new strand synthesis): DNAP III holoenzyme

DNA Replication-- Elongation

Addition of DNA polymerase III holoenzyme forms a replisome

Primers must be occasionally laid down on the lagging strand to prime Okazaki fragment synthesis. This is done by the DnaG primase which occasionally reassociates with the DnaB helicase to lay down a new primer on the lagging strand.

Leading strand

Leading strand

DNA Replication-- Elongation

DNA Replication-- Elongation

DNA Replication-- Elongation

A “snapshot” of DNA replication

DNA Replication-- Elongation

DNA Replication-- Elongation

Remove the primers and ligate Okazaki fragments: (DNAP I + Ligase)

DNA Replication-- Elongation

（ 1 ）除去引物： DNA polI ，有 5′→3′ 核 酸外切酶的活性，可把 RNA 引物除去， 所出现的缺口（ gap ）由 DNA polI 按 模板要求将缺口填满。

（ 2 ） DNA 连接酶将相邻的两个核苷酸的磷 酸二酯键连接起来成大分子 DNA ，再 形成一定的空间结构。

DNA Replication-- Elongation

• Terminate replication: Ter (termination sequence)+ Tus (termination utilization substance)

• Separate Daughter DNAs: DNA Topo IV

DNA Replication-- Termination and Separation

Let’s go over the details of DNA Replication:

Three steps

DNA Replication--Summary

1) Initiation （起始）Find the originUnwind the helix2) Elongation （延伸）Synthesize primers ElongateRemove the primers and ligate Okazaki fragments3) Termination and Separation （终止与分离）Terminate replication Separate Daughter DNAs

DNA Replication--Summary

DNA　Replication　is　an　Ordered　Series　of　Steps.

Could　you　tell　me　the　order　of　enzymes?　

DNA Replication--Summary

• Topoisomerase （拓扑异构酶）• DNA Helicase （ DNA 解链酶）• Single-stranded DNA binding proteins （ SSB ，

单链结合蛋白）• Primase （引发酶）• DNA –dependent DNA polymerase III (DNA 聚合

酶 III ）• The Enzymes responsible for removing RNA

primers (DNA polymerase II))•   DNA ligase （ DNA 连接酶）

DNA Replication--Summary

DNA Synthesis is More Complex in Eukaryotes 　 ( 真核 ) Than in Prokaryotes 　 ( 原核 )

Eukaryotic DNA Replication Like E. coli, but more complex

Chromatin and Nucleosome Multiple origins of replication DNA replication occurs just at S phase of the cell

cycle and is controlled by many proteins Okazaki fragments are shorter than in ProkaryotesReplication forks run a slower speed than in

ProkaryotesTwo rounds of replication cannot occur at the same

timeTelomerase is required

真核生物 DNA 复制与原核生物 DNA 复制大体相同，但有差异。

（ 1 ）原核生物每时每刻都在复制，而真核生物 DNA 的复制 在细胞周期的 S 期。 （ 2 ）原核生物 DNA 的复制只有 1 个复制起始点，而真核生 物 DNA 的复制有许多复制起始点。 （ 3 ）原核生物与真核生物 DNA 复制的酶不同，切除引物的酶 亦不同。（ 4 ）真核生物的 DNA 与组蛋白组装成核小体。

（ 5 ）端粒和端粒酶：真核生物染色体 DNA 是双链线状，由于 DNA 合成需要 RNA 作引物，引物除去后， 5′ 端留下一 段无法填补的空缺，这样 DNA 复制后就会愈来愈短，而 生物体有端粒酶来解决这个问题。

High Fidelity of Replication

Balanced levels of dNTPs.High selectivity of DNAPs based on

Watson-Crick base pairing (to the template base)

Proofreading of DNAPs by means of their 3' -->5' exonuclease.

Mismatch repair system.RNA primers are removed by highly

accurate Pol I enzyme.

DNA polymerase error rates

• Initial pairing error = 1/105

• After proofreading = 1/107~1/108

• mismatch repair = 1/1010~1/1011

• Human genome = 3.2 x 109 bp

• ~3 errors/replication!

The　following　is DNA　Damage　and　Repair

DNA Damage and Repair

• DNA is the only biomolecule that is specifically repaired. All others are either degraded or replaced.

• >100 genes participate in various aspects of DNA repair, even in organisms with very small genomes.

• Many, perhaps most, cancers are at least partially attributable to defects in DNA repair.

Types of Repair• Direct repair

Photolyase （光裂解酶） & Guanine Methyl transferase

（鸟嘌呤甲基转移酶）• Excision repair

1) Base excision 2) Nucleotide excision

3) Mismatch repair

• Damage tolerance

Attempts to minimize the effects of damage

that has not been repaired.

SOS repair & Recombinational repair

Photolyase contains two chromophores that absorb light energy. In all photolyases, one of the chromophores is FADH-, and the other is either methenyl-tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaflavin (8-HDF). MTHF and 8-HDF act as primary light gatherers (green in the diagram), transferring their energy to FADH- (yellow). The energy from FADH- is used to split the dimer

1. Removal of the incorrect base by an appropriate DNA N-glycosylase to create an AP site

2. Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3'-OH terminus adjacent to the APsite

3. Excision of the AP site, followed by extension of the 3'-OH terminus by a DNA polymerase

Steps in Base Excision Repair

A DNA glycosylasebinds to damaged base

AP endonuclease binds and cleaves the DNA backbone

Exonuclease removes a segment of DNA

DNAPI fills in the gap

Ligase seals the nick

Cleaves N-glycosidic bondNote that the AP siteIs identical to that made by spontaneousdepurination or depyrimidination.

DNA Glycosylases

The crystal structure of human uracil DNA glycosylase is shown bound to a DNA helix.

• 2 UvrA proteins form a complex with one UvrB protein in an ATP-dependent reaction

• The complex recognizes UV damage by the bend in the helix

• The UvrA proteins dissociate from the complex after ATP hydrolysis.

• This leaves UvrB bound across from the damage

Nucleotide Excision Repair in E. coli

• Now UvrB can recruit UvrC protein to the complex

• UvrC activates UvrB to nick the DNA 4 nts 3’ from the pyrimidine dimer

• Then UvrB activates UvrC to nick the DNA 7 ntds 5’ from the pyrimidine dimer

• This leaves a fragment of DNA containing the damage that can now be removed

Nucleotide Excision Repair in E. coli

• A helicase, UvrD, uses ATP hydrolysis to power the unwinding of the damaged DNA fragment. This reomoves UvrC

• The gap in the DNA is now filled in by DNAPI or II, reomving uvrB in the process

• Finally, DNA ligase seals the nick

Nucleotide Excision Repair in E. coli

Repair of Mismatch Replication ErrorsRepair of Mismatch Replication Errors(in E. coli)(in E. coli)

1.

2.

1. MutSMutS recognizes mismatches and binds to them. Binding of MutLMutL stabilizes the complex.

2. The MutS-MutL complex activates MutHMutH, which locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group.

Continued from the previous slide

2.

3.

4.

3. A helicase (UvrDUvrD) unwinds from the nick in the direction of the mismatch, and a single-strand specific exonuclease cuts the unwound DNA

4. The gap is filled in by DNAP III and sealed by DNA ligase.

SOS Repair-Error-prone replication

The SOS repair system is induced in response to major damage to the bacterial DNA or in response to agents which inhibit DNA replication. The system is a complex one with over 20 genes involved. Two of these are the important regulator genes: lexA and recA.

LexA is a repressor （阻遏蛋白） that regulates the expression of all of the other SOS repair genes, including recA. It also regulates its own synthesis. LexA is a dimer. Each monomer has a DNA binding domain and a dimerization domain, however, the protein will not bind to DNA unless it has formed a dimer first. Normally, LexA binds to its operators （操作子） to block expression of the SOS repair genes.

Recombinational Repair

Also known as post-replication repair, this system permits the cell to tolerate damage without actually repairing it. It depends on the mechanisms of homologous recombination （同源重组） to replace a damaged region of DNA that cannot be repaired with a good copy of the same region.

A DNA molecule has a T dimer

this molecule is being replicated. A PolIII will be unable to correctly copy the T dimer

DNA Pol　II reinitiates DNA synthesis downstream of T dimers.

This cannot be repaired by the usual repair systems. However, the exposed ssDNA with the correctly synthesized daughter molecule

One daughter molecule still contains the T dimer but the opposite strand has the correct sequence. The other daughter now contains a gap but this gap can be repaired correctly by the usual repair systems

DNA 修复

DNA 复制的速度很快，每分子酶每分钟合成约 1000 个核苷酸片段。 E.Coli 5×106bp, 30min;

human 3×109bp, 8h 。另外生物体生长的环境中种种物理和化学因素可作用于 DNA ，引起 DNA 结构的改变，使 DNA 受到损伤（ damage ），生物体有修复系统可使受损伤的 DNA 得到修复。

（ 1 ）光复活作用（ photoreactivation ） :

光复活酶（ photoreactivating enzyme ）

1. 损伤：

形成嘧啶二聚体

2. 形成酶－ DNA 复合物：

光复合酶结合于损伤部位

3. 酶被可见光所激活

4. 修复后释放酶

5' 3'

5' 3'

hυ

（ 2 ）切除修复（ excision repair ）

（ 3 ） 重组修复（ recombination repair ）

（ 4 ）错配修复（ mismatch repair ）

错配修复实际上是一种特殊的核苷酸切除修复，

它专门用来修复在 DNA 复制中出现的新合成 DNA

链上的错配的碱基。但如何避免将 DNA 链上正确

的碱基切除呢？这就需要将 old strand 和 new

strand区分开来。

原核生物利用甲基化区分 old strand 和 new

strand ，因此原核细胞中的错配修复也称甲基化指导的错配修复（ methyl-directed mismatch repair ）。原核细胞 DNA 的甲基化位点位于 5′GATC序列中腺嘌呤碱基的 6 位 N 原子上，催化甲基化的酶为 Dam

甲基化酶（ Dam methylase ），刚合成的 DNA 新链上的 GATC序列还没有来得及甲基化，而作为模板的 old strand早已被甲基化了，利用这种差别区分old strand 和 new strand 。

一旦发现错配碱基，即将未甲基化的链切除，并以甲基化的链为模板进行修复合成。

错配修复是一个非常耗能的过程。错配的碱基距离 GATC序列越远，被切除的核苷酸就越多，重新合成新链所需要消耗的脱氧核苷三磷酸单体就越多。无论消耗多少 dNTPs ，目的都是为了修复一个错配的碱基，这说明机体为了维护遗传物质的稳定性可以说不惜一切代价。

真核生物 DNA 错配修复机制与原核生物大致相同，但区分 old strand 和 new strand 的机制不同，详细的机理还不十分清楚。

(5). SOS 修复（ SOS repair ）：

SOS 比喻细胞处于危险状态，是细胞 DNA 合成受到阻断或 DNA 受损伤时诱导产生的一种错误倾向修复。在 DNA 合成受阻或 DNA 受损伤时诱导出一种新的 DNA 聚合酶，这种新的 DNA 聚合酶能通过 DNA 损伤部位而进行复制，但复制的精确度很低。校对功能很差，因而容易出现复制的差错，从而导致高的突变率。

四、 DNA 复制的高度忠实性 DNA 的复制是高度忠实的，出现差错的机会很小，出错的几率在 10-8 ～ 10-10之间，即每复制108 ～ 1010bp才出现 1次错误。主要有 5 种机制使DNA 复制的错误率降到很低。

2. DNA 聚合酶的两步反应机制。 3. DNA 聚合酶具有自我校对能力，可及时切除错配的碱基。 4. 错配修复。 5. RNA 引物。

1. 通过核苷酸合成的调节机制保持细胞内 4 种脱氧核苷 三磷酸浓度的平衡。