Chapter 10

DNA Replication

Function of DNA:

DNA is the carrier of genetic information in chromosome.

DNA replication is accord with cell division in vivo

Parent cell

Daughter cell

Daughter cell

Each daughter cell obtain a newly genetic information which is identical of parent cell during cell division.

Section 1

General Concepts of DNA Replication

The structure and function of DNA:

• Structure of DNA:DNA is composed of two complementary strands of polynucleotides.

Antiparallel5’

3’5’

3’

DNA replication

• Replication: It is a process in which genetic information is transmitted from parental DNA to daughter DNA. During it process to form two newly dsDNA which is identical of parental DNA .

replication

parental DNAdaughter DNA

Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

§1.1 Semi-Conservative Replication

• Conception: The original double-stranded DNA opens up and both strands serve as template for the synthesis of new DNA, The products of the reaction are two daughter double stranded DNA molecules. each of them has one original template strand and one newly synthesized strand DNA.

Semiconservative replication

§1.1 Semi-Conservative Replication

Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N medium

The first generation

grow in 14N medium

The second generation

§1.2 Bidirectional Replication• Bi-directional: replication is started in a

single origin C site , extending in two direction , It process is called bi-directional replication.

Replication fork :• During the initiation, the double

helix opens up , and both single strands served as template for synthesis of new DNA , forms “Y” shape , called replication fork .

3'

5'

5'

3'

5'

3'

5'3'

direction of replication

Replication fork

Replication of prokaryotes

The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.

Replication of eukaryotes

• Chromosomes of eukaryotes have multiple origins.

• The space between two adjacent origins is called the replicon, a functional unit of replication.

origins of DNA replication (every ~150 kb)

§1.3 Semi-continuous Replication

The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized only from the 5´ end to the 3´end.

leading strand and lagging strand If it synthesis direction of the daughter strand

accord with the replication fork shift direction, new DNA is made in a continuous piece in the correct 5ˊ-3ˊ direction , called leading strand .

• On the other daughter strand, it synthesis direction is against the replication fork shift direction. DNA polymerase synthesizes many short pieces of new DNA in 5ˊ-3ˊ direction , and joins these pieces together by ligase . The new strand which is made by this discontinuous method is called lagging strand .

• In the process of replication , the synthesis

of leading strand is continuous, in lagging stra

nd , DNA polymerase synthesize short pieces o

f new DNA in 5-3 direction , and then joins the

m together . The discontinuous fragments in la

gging strand are called okazaki fragment .

Okazaki fragments

Semi-continuous replication

3

5

3

5Fork move direction

3´

5´

3´

3´

5´ Fork shift dir

ection

5

Leading strand

Lagging strand

Synthesis direction of leading strand accord with the replication fork shift direction, and synthesis direction of lagging strand is against the replication fork shift direction.

Template: double stranded DNA

Substrate: dNTP

Primer: short RNA fragment with a free 3´-OH end

Enzyme: DNA-dependent DNA polymerase (DDDP),

other enzymes,

protein factor

DNA replication system

Section 2

Enzymology

of DNA Replication

• The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in 1959.

• Later, DNA-pol II and DNA-pol III were identified in experiments by mutated E.coli cell line.

§2.1 DNA Polymerase

DNA-pol of prokaryotes

• All of DNA Polymerase possess the following biological activity.

1. 53 polymerizing(for replication)

• Function: Recognizes the deoxynucleotide on the DNA template and then adds a complementary dNTP to the 3’-OH of the primer ,creating a 3’,5’phosphodiester bond on the daughter strand.

2. exonuclease (for proofreading)• Function: Check the base pair between parent

and daughter strand starting from the end of DNA,and remove mismatched deoxynucleotide from daughter strand.

3´→5´ exonuclease activity excise mismatched

nuleotides

C T T C A G G A

G A A G T C C G G C G

5' 3'

3' 5'

Exonuclease functions

The function of different DNA-pol of prokaryotes

• Polymerase : Single peptide chain, the Ⅰfunction is proofreading and repairing .

• polymerase :Only has its activity Ⅱ without DNA-pol and .Ⅰ Ⅲ

• polymerase :It is the Ⅲ most effective polymerase in synthesis of new strand DNA.

Klenow fragment

• Proteinase hydrolyze polymerase into two fragⅠment, larger one and smaller one .The larger one has the activity of DNA polymerase activity and 3’→5’exonuclease activity, also called Klenow fragment ,which can be use as “tool enzyme” in molecular study.

N end C end

caroid

DNA-pol Ⅰ

DNA-pol of eukaryotes

DNA-pol : elongation DNA-pol III

DNA-pol : primase and extend the lagging strand

DNA-pol : replication with low fidelity

DNA-pol : polymerization in mitochondria

DNA-pol : proofreading and filling gap

DNA-pol I

DNA-pol II

§2.2 Primase

• The replication is initiated by the formation of a short RNA (approximately five nucleotides long) which served as the primer for DNA polymerize. The synthesis of this short fragment RNA was catalized by primase , using DNA as template .

• Attention:It’s very special phenomenon during DNA replication.

The initiation phase of DNA replication

§2.3 Helicase

• Unwinding of double helix DNA is required before replication, because the template must paired with its complement dNTP. Helicase can use the ATP to unwind double helix DNA forming a replication fork.

§2.4 SSB protein

SSB(single-stranded DNA binding protein):

When DNA was unwinded into two single strand by helicase, it intend to form double-stranded DNA again.SSB binds to single strand DNA and keep the single state of DNA in replication.

§2.5 Topoisomerase• During unwinding double helix DNA, the

downstream double helix DNA over wrapped.

• The superhelix template needs to be released by topoisomerases.

There are two kinds of topoisomerase:

• Top I : It can break phosphodiester bond of just one strand of the duplex and then rejoin it, thus unwind supercoiled DNA.

• Top II : DNA topoisomerase break the double helix , unwind supercoiled DNA.

§2.6 DNA Ligase

• During replication , the synthesis of new strand may be discontinuous , to form many okazaki fragment . the DNA ligase can link two adjacent fragment by 3’-5’phosphodiester bond ,form the integrated daughter strand.

Section 3

DNA Replication Process

• Initiation: recognize the origine point, separate dsDNA, primer synthesis, …

• Elongation: add dNTPs to the existing strand, form phosphodiester bonds, correct the mismatch bases, extending the DNA strand, …

• Termination: stop the replication.

Replication is a continuous process , to describe it clearly , we separate it into three stage :

• The replication starts at a particular point called origin.

• The origin of E. coli is 248 bp long and AT-rich.

§3.1 Replication of prokaryotes

a. Initiation

Dna ADna B Dna C

DNA topomerase

5'3'

3'

5'

primase

Primosome complex

SSB

5' 3'5'

dATPdGTP

dTTPdCTP

dTTPdGTP

dATPdCTP

OH 3'3'DNA-pol

目 录

• dNTPs which is complementary to template are continuously connected to the primer or the nascent DNA chain by DNA-pol III.

• The nature of the chain elongation is the series formation of the phosphodiester bonds.

b. Elongation

• Because of the circular structure of chromosome,the replication of prokaryote is bidirectional from one origin, and the two replication forks must meet at opposite point.

• All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase.

c. Termination

§3.2 Replication of Eukaryotes

• DNA replication is closely related with cell cycle.

• Multiple origins on one chromosome, and replications are activated simultaneously.

Cell cycle

• The eukaryotic origins are shorter than that of E. coli.

• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity).

• Needs topoisomerase and replication factors (RF) to assist.

Initiation

• DNA replication and nucleosome assembling occur simultaneously.

• Overall replication speed is compatible with that of prokaryotes.

b. Elongation

3'

5'

5'

3'

3'

5'

5'

3'

connection of discontinuous

3'

5'

5'

3'

3'

5'

5'

3'

segment

c. Termination

• The terminal structure of eukaryotic DNA of chromosomes is called telomere.

• Telomere is composed of terminal DNA sequence and protein.

• The sequence of typical telomeres is rich in TTAGGG repeat sequence.

• The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.

Telomere

• The eukaryotic cells use telomerase to maintain the integrity of DNA telomere.

• The telomerase is composed of

telomerase RNA telomerase association protein telomerase reverse transcriptase

• It is able to synthesize DNA using RNA as the template.

Telomerase

Inchworm model

• Telomerase may play important roles in human aging and in cancer cell biology .

Significance of Telomerase

Section 4

Reverse Transcription

§4.1 Reverse Transcription• Reverse transcription: is a process in whic

h genetic information is transmitted from RNA to DNA . It’s phenomenon often occure in the process that eukaryote cell be infected by RNA virus.

• In these RNA virus,the genetic information carrier is ssRNA instead of dsDNA (such as ssRNA viruses,HIV virus or tumour virus).

Viral infection of RNA virus

During RNA virus infect Host cell,the genetic information must be transmitted from ssRNA to ds DNA ,and integrated into the chromosome of Host cell.Then replicate and transcribe with the DNA from Host cell.

Process of Reverse transcription

• Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.

• Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.

• Synthesis of the second ssDNA using the left ssDNA as the template, forming a dsDNA.

Significance of RT

• RNA plays a key role just like DNA in the genetic information transfer and gene expression process.

• RNA could be the molecule developed earlier than DNA in evolution.

• Reverse transcription has become a extremely important tool in molecular biology to select the target genes.

Section 5

DNA Damage and Repair

Mutation is a change of nucleic acids in genomic DNA of an organism. The mutation could occur in the replication process as well as in other steps of life process.

§5.1 Mutation

§5.2 Causes of Mutation

DNA damage

UV radiation

viruses

carcinogensPhysical factors

evolution

infection

T

G

spontaneous mutation

Chemical modification

N

N O

O

CH3

R

PN

N O

O

CH3

R

N

N O

O

CH3

R

P

N

N OR

UV

O

CH3

( T T )

)

Mutation caused by chemicals

• Carcinogens can cause mutation.

• Carcinogens include: • Food additives and food preservative

s; spoiled food

• Pollutants: automobile emission; chemical wastes

• Chemicals: pesticides; alkyl derivatives; -NH2OH containing materials

Point mutation is referred to as the single nucleotide alternation.

a. Point mutation (mismatch)

§5.3 Types of Mutation

C T T C A G G A

G A A G T C C G G C G

5' 3'

3' 5'

HbS HbA

chains CAC CTC

mRNA GUG GAG

AA residue 6 in chain Val Glu

Hb mutation causing anemia

Single base mutation leads to one AA change, causing disease.

http://baike.baidu.com/image/027a45b5ddc03b8e36d3cacf

b. Deletion and insertion

• Deletion: one or more nucleotides are

deleted from the DNA sequence.

• Insertion: one or more nucleotides are inserted into the DNA sequence.

Deletion and insertion can cause the reading frame shifted.

Frame-shift mutation

Normal

5´… …GCA GUA CAU GUC A… …

Ala Val His Val

Deletion C

5´… …GAG UAC AUG UCA … …

Glu Tyr Met Ser

c. Rearrangement

It is the exchang or transfer of genetic information between homologous chromosome ,resulting in the formation of new characteristics not found in either parental DNA.

• DNA repairing is a kind response made by cells after DNA damage occurs, which may resume their natural structures and normal biological functions.

§5.4 DNA Repairing

N

N O

O

CH3

R

PN

N O

O

CH3

R

N

N O

O

CH3

R

P

N

N OR

UV

O

CH3

( T T )

)

Light repairing

Excision repairing

Recognise and cleave injured fragment by indonuclease

• One of the most important and effective repairing approach.

• UvrA and UvrB: recognize and bind the damaged region of DNA.

• UvrC: excise the damaged segment.

• DNA-pol Ⅰ: synthesize the DNA segment to fill the gap.

• DNA ligase: seal the nick.

Excision repairing

Recombination repairing

• It is used for repairing when a large segment of DNA is damaged.

SOS repairing

• It is responsible for the situation that DNA is severely damaged and the replication is hard to continue.

• If workable, the cell could be survived, but may leave many errors.