DNA Replication

DNA DNA

DNA: Double helix and antiparallel 5 3

3 5 Semiconservative Meselsson & Stahl Double helix DNA must be separated Leading strand vs. Lagging strand Okazaki fragments In E.coli start point: OriC (Origin of replication)

DNA Replication
Required enzymes: - DNA polymerase I and III - RNA primase (Primosome complex) - DNA helicase - DNA ligase

- DNA gyrase
Other proteins: - Single stranded binding protein (SSBP)

- Initiator protein (dnaB)

Primer RNA

 Replication orientation: 5 3
Leading strand
(continous)

Movement of Replication fork

5
3

Okazaki fragment

Lagging strand
(discontinous)

Replication fork

Okazaki Fragments In E. coli: 1000-2000 nt

Joined by DNA ligase

 Unwinding of DNA
DNA helicase

- catalyze unwinding process of helix DNA - separate double stranded DNA - 2 types in E.coli: Helicase II (lagging strand) & Rep protein (leading strand) - require ATP
5 3

Rep protein

Helicase II

Leading Lagging strand strand

 Single-stranded DNA binding protein = SSBP)

- Bind tightly to DNA - Stabilize separation of double stranded DNA as template - no requirement of ATP

SSBP

 DNA gyrase (Topoisomerase II)

- Catalyze the synthesis of negative supercoil of DNA - Essential for unwinding process - Require ATP
Primer RNA - Initiate the synthesis of DNA - Synthesis of RNA primer is catalyzed by primase & RNA polymerase

Primase

-6 other proteins Primosome

- BM 60 kD - Initiate the synthesis of Okazaki fr. (lagging strand) - Synergistic interaction with RNA polymerase initiate the synthesis of leading strand

3 5 5

Primer RNA
3

3
5 3

- Length of primer RNA depends on species ca. 1-60 nt E. coli: 10-60 nt

-After DNA synthesis began primer will be digested

DNA polymerase I

- Isolated in E.coli by Arthur Kornberg (1957)

- Single polypeptide, BM 103 kD - Functions: 1. Polymerization (adding nt to 3-OH end of DNA) (DNA) n + dNTP (DNA) n+1 + PPi

Polymerization reaction Required components: - Precursor: dNTP (dATP, dGTP, dCTP, dTTP) - Mg2+ - Primer RNA (ujung 3-OH bebas) - template DNA Orientation of polymerization reaction: 5 3

A
5

G P P

C
3

dGTP OH

PPi
5

A P

G P

G
3

OH

DNA Polymerase I *Addition of base complementary to template * Synthesize only short DNA 20 nt * Rate of synthesis: 10 nt/second

2. DNA Repair * Exonuclease activity: 3 5 - Separate the false nucleotide in replication - Proof read mechanism by DNA polymerase I DNA replication very accurate

TA
TA TA

Exonuclease 3 5

A
5

OH

* Exonuclease activity 53 - Separate up to 10 nt from 5-end of single stranded DNA

5 Exonuclease 53
C
C C

A
A A TA

(nick)

exonuclease 53
3

TA
TA

- Repair false nucleotide in double stranded DNA - Role: Repair of mutation caused by UV irradiation & chemical mutagene Digest primer RNA

DNA polymerase I
small fragment
N

large fragment (fr. Klenow)

exonuklease 35 polymerase
C

exonuklease 53

DNA polymerase III - DNA replication - BM 900 kD -H oloenzyme, 10 subunit protein 5 subunit core enzyme

- Functions:

Polimerization 5 3 * Subunit * DNA synthesis up to thousands nt * Synthesis rate: 1000 nt/second

Exonuclease 3 5 * Subunit * Editor for DNA replication accuracy of replication increase up to 200 x

Dimer komples leading & lagging strand are synthesized simultaneously by a dimer complex DNA pol. III enzyme

leading strand

Cetakan lagging strand melengkung ke belakang, melingkari enzim tsb sehingga enzim tsb dpt melakukan polimerisasi pd orientasi yg sama dgn cetakan leading strand Setelah fr. Okazaki terbentuk lengkap lengkungan (loop) melonggar & lepas dr enzim tsb cetakan lagging strand yg berikutnya (belum berpasangan) melingkari enzim pembentukan fr. Okazaki berikutnya

D. DNA ligase - Bind fr. Okazaki - Catalyze the synthesis of phosphodiester bonds between 3-OH end of one DNA and 5-P end of the other DNA

- Require energy from hydrolysis:

NAD+ NMN+ + AMP (E. coli) ATP PPi + AMP ( Eukaryot, bakteriophage T4) - Reactions:
O DNA-3-OH +
-O-P-O-5-DNA

DNA ligase + ATP atau NAD+

O DNA-3-O-P-O-5-DNA O-

O-

Termination
- In locus Ter (T) - Lokus Ter contains of GTGTGTTGT sequences bind to Tus protein termination of DNA synthesis - Tus protein binds to Ter inhibition of DnaB helicase

OriC
Ter E Ter D Ter A

Ter F

Ter C Ter B

1. Replication runs through Ter E, Ter D, Ter A

and stops at Ter C or Ter B or Ter F

2. Replication runs through Ter F, Ter B, Ter C and stops at Ter A or Ter D or Ter E
replikasi berlawanan arah jarum jam replikasi searah jarum jam

Protein Tus

daerah terminasi

Protein Tus