dna replication.pdf

8/18/2019 DNA Replication.pdf

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1. Semiconservative


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2. Conservative


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3. Dispersive


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DNA ReplicationDNA Replication

DNA DNA

DNA: Double helix and antiparallel 5’ → 3’

3’ ← 5’

Semiconservative → eselsson ! Stahl

Double helix DNA must be separated

"eadin# strand vs. "a##in# strand

$%a&a%i 'ra#ments

(n ).coli start point: OriC (Origin of replication)


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DNA ReplicationDNA Replication

*e+uired en&,mes: - DNA pol,merase ( and (((

- *NA primase /rimosome complex0

- DNA helicase

- DNA li#ase

- DNA #irase

$ther proteins: - Sin#le stranded bindin# protein

SS/0

- (nitiator protein dna0

/rimer *NA


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*eplication orientation: 5’ → 3’

ovement o' *eplication 'or%

3’

5’5’3’

3’5’

3’

5’

$%a&a%i 'ra#ment

Leading strand (continous)

Lagging strand

(discontinous)*eplication 'or%

$%a&a%i ra#ments

(n ). coli: ± 1444-2444 nt in eu%ariot: 144-244 nt

6oined b, DNA li#ase


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7n8indin# o' DNA

DNA helicase

- catal,&e un8indin# process o' helix DNA - separate double stranded DNA

- 2 t,pes in ).coli: 9elicase (( la##in# strand 0 ! *ep protein leadin# strand 0

- re+uire A/

Rep protein elicase !!

3’

3’

5’

5’

"eadin#

strand

"a##in#

strand


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Sin#le-stranded DNA bindin# protein ; SS1/ - ind ti#htl, to DNA

- Stabili&e separation o' double stranded DNA as

template - no re+uirement o' A/

3’5’

3’ 5’

SS"#


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DNA #irase opoisomerase ((0

- Catal,&e the s,nthesis o' ne#ative supercoil o' DNA- )ssential 'or un8indin# process

- *e+uire A/

/rimer *NA

- (nitiate the s,nthesis o' DNA

- S,nthesis o' *NA primer is catal,&ed b,

primase ! *NA pol,merase


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Primase

-< other proteins /rimosome

-


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- "en#th o' primer *NA depends on species ca. 1-


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/ol,meri&ation reaction *e+uired components:

- /recursor: dN/ dA/ d/ dC/ d/0

- #2@

- /rimer *NA uBun# 3’-$9 bebas0 - template DNA

$rientation o' pol,meri&ation reaction: 5’ → 3’

/ / / / / $9$95’ 5’

3’3’

A G C A G C GdGTP PPi


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DNA #ol$merase !

Addition o' base → complementar, to template

S,nthesi&e onl, short DNA 24 nt

*ate o' s,nthesis: 14 ntEsecond

2. DNA *epair

)xonuclease activit,: 3’ → 5’

- Separate the 'alse nucleotide in replication

- /roo' read mechanism b, DNA pol,merase (

DNA replication ver, accurate


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F A

F A

F A

C A

$9

9idrolisis olehe%sonu%lease 3’ 5’

3’

3’5’

5’


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)xonuclease activit, 5’3’

- Separate up to 14 nt 'rom 5’-end o' sin#le stranded

DNA

Hydrolysis by 5’3’ exonuclease

(nick )

C A

C A

C A

F A

F A

F A

Hydrolysis by 5’3’

exonuclease

- *epair 'alse nucleotide in double stranded DNA

- *ole:

♥ *epair o' mutation caused b, 7G irradiation !

chemical muta#ene

♥ Di#est primer *NA

5’

5’3’

3’


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DNA pol$merase !

small fragment large fragment (fr% &leno')

eonulease eonulease pol$merase

5’

3’ 3’

5’

N C

DNA pol$merase !!!

- DNA replication- >44 %D

-9 oloen&,me H 14 subunit protein ? subunit core en&,me


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- unctions:

* /olimeri&ation 5’ 3’

Subunit

DNA s,nthesis up to thousands nt

S,nthesis rate: 1444 ntEsecond

* )xonuclease 3’ 5’ Subunit

)ditor 'or DNA replication accurac, o' replication

increase up to 244 x


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- *eactions:

$ $

DNA-3’-$9 @ +$-/-$-5’-DNA DNA-3’-$-/-$-5’-DNA

$+ $+

D. DNA ligase

- ind 'r. $%a&a%i

- Catal,&e the s,nthesis o' phosphodiester bondsbet8een 3’-$9 end o' one DNA and 5’-/ end o' theother DNA

- *e+uire ener#, 'rom h,drol,sis:

I NAD@ NN@ @ A/ ). coli0

I A/ //i @ A/ )u%ar,ot ba%teriopha#e J0

, A-# atauNAD,

DNA ligase


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Dimer %omples leadin# ! la##in# strand are s,nthesi&edsimultaneousl, b, a dimer %omplex DNA pol. ((( en&,me

he la##in# strand template is looped around and each

replicative DNA pol,merase moves 5’ 3’ relative to itsstrand cop,in# template and s,nthesi&in# a ne8 DNAstrand


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A'ter $%a&a%i 'ra#ment has been completel,s,nthesi&ed the loop is loosened and the en&,me8ill move to s,nthesi&e another 'ra#ment $%a&a%i.


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-ermination

- (n locus :er 0

- er locus contains o' se+uences

bind to us protein termination o' DNA s,nthesis - us protein binds to er inhibition o' Dna helicase

OriC

-er .

-er D

-er A

-er C

-er / -er "


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-us protein -us protein

Countercloc%8ise Cloc%8ise

e

r

B

T

e

r

T

e

r

e

r

D

1. *eplication runs throu#h er ) er D er A

and stops at er C or er or er

2. *eplication runs throu#h er er er C

and stops at er A or er D or er )

dna replication.pdf

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