Septelia Inawati WanandiDept. of Biochemistry and Molecular Biology FKUI

DNA Replication 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 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’

5’5’

3’

3’5’

3’

5’

Okazaki fragmentLeading strand(continous)Lagging strand(discontinous)

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

Rep protein

Helicase II

3’

3’

5’

5’

Leadingstrand

Laggingstrand

Single-stranded DNA binding protein = SSBP) - Bind tightly to DNA - Stabilize separation of double stranded DNA as template - no requirement of ATP

3’5’

3’ 5’

SSBP

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

3’3’

3’5’5’

5’5’

3’Primer RNA

- 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’

P P P P P OHOH5’ 5’

3’3’

A G C A G C G dGTP PPi

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

T … A T … A T … A C A

OH

Exonuclease 3’ 5’

3’

3’5’

5’

* Exonuclease activity 5’3’ - Separate up to 10 nt from 5’-end of single stranded

DNAExonuclease 5’3’

(nick)

C A C A C A T … A T … A T … A

exonuclease 5’3’

- Repair false nucleotide in double stranded DNA - Role: Repair of mutation caused by UV irradiation &

chemical mutagene Digest primer RNA

5’

5’3’

3’

DNA polymerase I small fragment large fragment (fr. Klenow)

exonuklease exonuklease polymerase 5’3’ 3’5’

N C

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

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

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

- Reactions: O O DNA-3’-OH + -O-P-O-5’-DNA DNA-3’-O-P-O-5’-DNA O- O-

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)

+ ATP orNAD+

DNA ligase

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 ETer DTer A

Ter CTer F Ter B

Protein Tus

Protein TusTerminati

on site

Bidirectional Replication

Ter B Ter C Ter A

Ter D

1. Replication runs through Ter E, Ter D, Ter A and stops at Ter C or Ter B or Ter F2. Replication runs through Ter F, Ter B, Ter C and stops at Ter A or Ter D or Ter E

CELL CYCLE

TELOMERASE

TELOMERASERibonucleoprotein 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)