Characteristics of replication Semi-conservative replication Bidirectional replication...

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Characteristics of replication Semi-conservative replication Bidirectional replication Semi-continuous replication

Transcript of Characteristics of replication Semi-conservative replication Bidirectional replication...

Page 1: Characteristics of replication Semi-conservative replication Bidirectional replication Semi-continuous replication.

Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

Page 2: 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.

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§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.

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Semi-continuous replication

3

5

3

5Fork move direction

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.

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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

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Section 2

Enzymology

of DNA Replication

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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.

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• 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.

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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

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The initiation phase of DNA replication

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§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.

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§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.

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§2.5 Topoisomerase• During unwinding double helix DNA, the

downstream double helix DNA over wrapped.

• The superhelix template needs to be released by topoisomerases.

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§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.

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Section 3

DNA Replication Process

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• 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 :

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• 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

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GATTNTTTATTT ··· GATCTNTTNTATT ··· GATCTCTTATTAG ···

53

E.Coli oriC

1 13 17 29 32 44

···TGTGGATTA-‖-TTATACACA-‖-TTTGGATAA-‖-TTATCCACA

58 66 166 174 201 209 237 245

Tandem Repeat Seq Inverted Repeat Seq

53

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Dna ADna B Dna C

DNA topomerase

5'3'

3'

5'

primase

Primosome complex

SSB

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Primase synthesis primerDnaG (dnaG)

cooperate DnaBDnaC (dnaC)

UnwindingHelicaseDnaB(dnaB)

Recognize ori CDnaA (dnaA)

protein Generic Name function

Replication initiation related Pr. in procaryote

SSB stabilize ssDNA

Topoisomerase unwind supercoiled DNA

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• The supercoil constraints are generated ahead of the replication forks.

• Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint.

• The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.

Releasing supercoil constraint

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目 录

• dNTPs which is complementary to template are cont

inuously connected to the primer or the nascent DN

A chain by DNA-pol III.

• The nature of the chain elongation is the series form

ation of the phosphodiester bonds.

b. Elongation

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5'

3'

3'

5'

5'

direction of unwinding3'

On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.

Leading strand

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Semi-continuous replication

3'

5'

5'3'

replication direction

Okazaki fragment

3'

5'

leading strand

3'

5'

3'

5'replication fork

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Replication Fidelity

• Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer.

• Enzymes use two mechanisms to ensure the replication fidelity.

– Proofreading and real-time correction

– Base selection

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• DNA-pol I has the function to correct the mismatched nucleotides.

• It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication.

Proofreading and correction

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• The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32.

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

c. Termination

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• Primers on Okazaki fragments are digested by RNase.

• The gaps are filled by DNA-pol I in the 5´→3´direction.

• The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by ligase.

Lagging strand synthesis

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Genome of E. coli

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3'

5'

5'

3'

RNAase

POH

3'

5'

5'

3'

DNA polymerase

P

3'

5'

5'

3'

dNTP

DNA ligase

3'

5'

5'

3'

ATP

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§3.2 Replication of Eukaryotes

• DNA replication is closely related with cell cycle.

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

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Cell cycle

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• 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

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• DNA replication and nucleosome assembling occur simultaneously.

• Overall replication speed is compatible with that of prokaryotes.

b. Elongation

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3'

5'

5'

3'

3'

5'

5'

3'

connection of discontinuous

3'

5'

5'

3'

3'

5'

5'

3'

segment

c. Termination

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• 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

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• 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

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Inchworm model

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

Significance of Telomerase

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Cell Cell divisiondivision

Cell divisionCell division

aging

telomerasetelomeraseTarget for Target for anti-canceranti-cancer

ImmortalizationImmortalization

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Section 4

Reverse Transcription

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§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).

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§4.1 Reverse Transcription

• The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses).

• The information flow is from RNA to DNA, opposite to the normal process.

• This special replication mode is called reverse transcription.

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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.

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Reverse transcription

Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.

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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 DNA-DNA duplex.

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Reverse transcriptase

Reverse transcriptase is the enzyme for the reverse transcription. It has activity of three kinds of enzymes:

• RNA-dependent DNA polymerase

• RNase

• DNA-dependent DNA polymerase

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Significance of RT

• An important discovery in life science and molecular biology

• 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.

• RT is the supplementary to the central dogma.

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Significance of RT

• This discovery enriches the understanding about the cancer-causing theory of viruses. (cancer genes in RT viruses, and HIV having RT function)

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

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Section 5

DNA Damage and Repair

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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

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§5.2 Causes of Mutation

DNA damage

UV radiation

viruses

carcinogensPhysical factors

evolution

infection

T

G

spontaneous mutation

Chemical modification

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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 )

)

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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

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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'

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• Transition: the base alternation from

purine to purine, or from pyrimidine t

o pyrimidine.

• Transversion: the base alternation be

tween purine and pyrimidine, and vis

e versa.

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Transition mutation

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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.

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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.

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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

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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.

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Gene Rrarrangement lead to mediterranean anemia

Hb β gene family on Ch

r 11

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

• DNA repairing is a supplementary to the proofreading-correction mechanism in DNA replication.

§5.4 DNA Repairing

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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

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Excision repairing

Recognise and cleave injured fragment by endonuclease

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• 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

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Recombination repairing

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

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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.