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DNA 的生物合成. Biosynthesis of DNA. The genetic central dogma. Ⅰ Concept of DNA replication or DNA biosynthesis. Semiconservative replication

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DNA 的生物合成

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DNA 的生物合成

Biosynthesis of DNA


The genetic central dogma


ⅠConcept of DNA replication or DNA biosynthesis

  • Semiconservative replication

  • The synthesis of a complementary DNA strand by forming phosphodiester linkages between nucleotides base-paired to template strand is catalyzed by large multienzyme complexes referred to as the DNA polymerases

  • The transmission of the genetic informations between DNA and DNA


parent DNA

strand

parent DNA

molecules

Daughter or new DNA

strand

daughter DNA

molecules

total conserve

commingle

semi conserve


  • The basic experiment condition

  • A experimental table

  • A centrifuge

  • A kind of bacterium

  • A few 14NH4Cl culture solutions

  • A few 15NH4Cl culture solutions

  • A few sucroses

7N14,15 / 1S22S22P3

Experimental basis of semiconservative replication


密度梯度实验


Significance of semiconservative

replication

TGCA

ACGT

TGCA

ACGT

TGCA

ACGT

Genetic conservativeness


ⅡEnzymes in DNA replication

  • DNA polymerases

  • DNA helicase

  • DNA topoisomerase

  • DNA single strand binding protein

  • Primase

  • DNA ligase


proof read, repair, filling

proof read, repair, filling

veriest replicase

substitute

substitute

major action

major action

in mitochondria

DNA polymerases

prokaryote

polymeras I

polymeras II

polymeras III*

eukaryote

polymeras *

polymeras 

polymeras

polymeras*

polymeras 


’

’

DNA polymerase III of E. coli


DNA polymerases function--1

5’3’ polymerase activity

5’

3’

N

N

N

+

OH3'

OH3'

5'

P

P

P

5'

P

P



5’

3’

N

N

N

OH3'

5'

P

P

P

+

PPi


DNA polymerases function--2

exonuclease activity

*

cut the fragment

of primer and

mutated

fragments

Polymerase I

Polymerase II

5’

5’

*

Polymerase I

Polymerase II

Polymerase III

Proof reading

C

A

3’

3’

G

G


high fidelity of DNA replication

(1) The strict base complementary

5’

5’

5’

5’

3’

3’

3’

3’

G

G

A

C

T

T

A

C


(2) the character of DNA polymerase selecting base

5’

T

A

A

A

T

T

A

A

T

T

A

A

T

T

T

T

The conformation of DNA

polymerase III is changeable,

as its affinity to nucleotide acids

G

3’

C

C

G

A

A

C

G

T

C

G

C

G

C

G

G

C

C

C

G

G

T


(3) The proof reading function of DNA polymerase

proof

reading

5’

5’

5’

5’

A

C

G

3’

3’

3’

3’

T

A

A

A

A


DNA helicase

Dna A, Dna B (rep), Dna C……Dna X

Dna T

Dna C

Dna B

Dna A

Dna C

Dna T


untwisting

ligate

cut

DNA topoisomerase

topoisomerase I:

breaks single strand DNA, not requires ATP

topoisomerase II:

breaks double strands DNA, requires ATP


DNA topoisomerase

normal helix

helicase

positive superhelix

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

DNA polimerase

1 2 3 4 5 6 7 8 9 10 11

topoisomerase

negative superhelix


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


The role of SSB

Dna B

SSB bank


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 factorshave binded

with DNA, then the primasehave binds with them and

formed a primosome at last.


the actions of primer and primase

Parental DNA template

5’

3’

SSB

primase

5’

3’

5’

3’

5’

3’

5’

3’

DNA polymerase III

5’

3’

3’

5’

New DNA strand


DNAligase

3’

5’

P

OH

P

OH

5’

3’

ATP

ADP+Pi

3’

5’

5’

3’


The compare of several enzymes, which

in formation of phosphodiester bond

enzymes

results

provides 3’-OH

provides 5’-P

1 DNA primer or dNTP (dNMP) n+1

polymerase extending

DNA strand

2 DNA no continued two single strands no continue

ligase  continue

3 DNA to break and put in order two put in order to DNA

topoisomerase DNA single strands superhelical structure

4 primase extending primer NTP primer


Ⅲ Process of DNA replication

  • Initiation of DNA replication

  • Extension of DNA replication

  • Termination of DNA replication


Initiation of DNA replication

Prokaryotic cell ( for instance E.coli)

1 A fixed origin (ori)

2 Bidirectional replication

Eukaryotic cell

a lot of origin


origin

theta

1

2

O

O

O

D

D

3’

21

2 1

21

21

3’

D

Direction

origin

O

O

Eukaryotic cell

Prokaryotic cell


leading strands

Helicase, SSB ...

Origin

2

1

lagging strands


The oriC of E coli.

GATCTNTTTATTT---GATCTNTTNTATT---GATCTCTTATTAC

55 66 166 174201 209 237 245

1 13 17 29 32 44

TGTGGATTA---TTATACACA------TTTGGATAA---TTATCCACA

palindrome structure

DnaT

DnaC

DnaB

DnaB

DnaA

DnaC

DnaT


topoisomerase

lagging strand

3’

polymerase III  subunit

5’

3’

single strand binding protein

3’

5’

polymerase III holoenzyme

leading strand

primer

5’

The extension of DNA replication


the termination of DNA replication

origin

0%

82%

32%

SV 40

E.coli

50%

terminate point

the terminate point of E.coli and SV40 virus


Parental DNA template strand

New DNA strand

the role of several enzyme in termination

of DNA replication

3’

5’

P

OH

3’

5’

RNA primer

3’

5’

degradation

P

OH

RNase

3’

5’

Pol I

filling

3’

5’

P

OH

3’

5’

ligation

Ligase

3’

5’

3’

5’


Chemical reaction in DNA replication

(dATP)m + (dCTP)n + (dGTP)y + (dGTP)z

(dAMP + dCMP + dGMP + dGMP) m +n+ y+z + (m + n + y + z) PPi


helicase

5’

3’

A C A G T G A T C A G A

5’

T G T C T C T

3’

primase

primase

H

O P P P

A

G

OH

single strand

binding protein

O

P

PP

HO

O

DNA polymerase Ⅲ

……dGTP/dTTP/dCTP/dTTP

RNase

DNA polymerase Ⅰ

ligase

O H 3’

5’

single strand binding protein

topoisomeraseⅡ


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