Septelia Inawati Wanandi

Dept. of Biochemistry and Molecular Biology FKUI

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’
Movement of Replication fork
3’ 3’
Leading strand
(continous) 5’ 3’
Okazaki fragment
3’ 5’
Lagging strand 5’
(discontinous) 5’
Replication fork

Okazaki Fragments
• In E. coli:  1000-2000 nt
• Joined by DNA ligase
Unwinding of DNA

DNA gyrase (Topoisomerase II)

- Catalyze the synthesis of negative supercoil of DNA
- Essential for unwinding process
- Require ATP
 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

3’ 5’
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

5’ 3’

SSBP

3’ 5’
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’ 3’
Primer RNA
3’ 5’
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 (3’-OH end)
- template DNA

 Orientation of polymerization reaction: 5’  3’

A G C A G C G
dGTP PPi
3’
3’
P P OH P P P
5’ OH
5’
 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 reading mechanism by DNA polymerase I
 DNA replication very accurate
 5’ 3’
T…A
T…A
Exonuclease 3’  5’
T…A
C A
3’
OH 5’
 * Exonuclease activity 5’3’
- Separate up to 10 nt from 5’-end of single stranded
DNA
5’ 3’
C A
Exonuclease 5’3’
C A
C A
T…A
exonuclease 5’3’ T…A
(nick)
T…A
3’ 5’
- 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 large fragment (fr. Klenow)

N C
exonuklease exonuklease polymerase
5’3’ 3’5’

DNA polymerase III

- DNA replication
- BM ± 900 kD
-H oloenzyme, 10 subunit protein
 5 subunit core enzyme
- Functions:
Ø Polimerization 5’  3’
* Subunit 
* DNA synthesis up to thousands nt
* Synthesis rate: 1000 nt/second
Ø Exonuclease 3’  5’
* Subunit 
* Editor for DNA replication accuracy of replication
increase up to 200 x
Dimer complex  leading & lagging strand are
synthesized simultaneously by a dimer complex DNA
pol. III enzyme

 By binding two replicative polymerases together and looping

the lagging strand  it can pass through the complex 
both strands can be made in one place

 DNA feeds through complex, rotating as it passes through 

the large protein complexes do not have to rotate around the
DNA
D. DNA ligase
- Binds to 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 (Eukaryotic cells, bakteriophage T4)

- Reactions:
O O
DNA ligase
DNA-3’-OH + -O-P-O-5’-DNA DNA-3’-O-P-O-5’-DNA
+ ATP or
O- NAD+ 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 C
Ter B
Ter F
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

Bidirectional
Replication

Protein Tus Protein Tus

Termination
site
CELL CYCLE
TELOMERASE
TELOMERASE
 Ribonucleoprotein complex containing a small RNA that
serves as a template for addition of a telomere
 Telomere: tandem repeats of a six nucleotide sequence
(TTAGGG  human)