HO Replikasi DNA

REPLIKASI DNA

Dr. Oeke Yunita, S.Si., M.Si., Apt. 1

Synthesis Phase (S phase)

• S phase during interphase of the cell cycle

• Nucleus of eukaryotes

2

Mitosis -prophase -metaphase -anaphase -telophase

G1 G2

S phase

interphase

DNA replication takes place in the S phase.

Handout Biologi Sel, Dr. Oeke Yunita

DNA Nucleotide

3

O=P-O O

Phosphate Group

N Nitrogenous base (A, G, C, or T)

CH2

O

C1 C4

C3 C2

5

Sugar (deoxyribose)

O


TAHAP – TAHAP

REPLIKASI DNA

1.Denaturasi (Pemisahan) untaian DNA

template

2. Inisiasi

3. Pemanjangan untaian DNA

4. Ligasi fragmen DNA

5. Terminasi

4 Handout Biologi Sel, Dr. Oeke Yunita

DNA Replication

• Begins at Origins of Replication

• Two strands open forming Replication Forks (Y-shaped region)

• New strands grow at the forks

5

Replication Fork

Parental DNA Molecule

3’

5’

3’

5’ Handout Biologi Sel, Dr. Oeke Yunita

Template: double stranded DNA

Substrate: dNTP

Primer: short RNA fragment with a

free 3´-OH end

Enzyme: DNA-dependent DNA

polymerase (DDDP),

other enzymes,

protein factor

DNA replication system


Origin of Replication Enzymes (Initiator Proteins) recognize the start site on template DNA and initially open up the DNA (single strands)

Helicases untwist the double helix at the replication forks

Primase begins DNA replication by laying an an RNA primer (10 nt long) for DNA polymerase to start at

Single-strand binding proteins bind to and stabilize the single-stranded DNA until it can be used as a template

DNA polymerase polymerizes new DNA in a 5’ to 3’ direction, proofreads the newly laden DNA, excises error ridden DNA and replaces that DNA with the correct sequence. Speed/fidelity: mutates 1 X 10-10

Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

Terminal replication proteins stall replication fork progress for polymeriation to catch-up

DNA ligase anneals nucleotides together, creating a DNA strand

Players involved in DNA Replication


I

III III

I

DNA



Replication of Strands

Handout Biologi Sel, Dr. Oeke Yunita 10

Replication Fork

Point of Origin

Components of the DNA Replication


Core proteins at the replication fork

Nature (2003) vol 421,pp431-435 12 Handout Biologi Sel, Dr. Oeke Yunita


DNA Primer synthesis On Lagging strand


Initiating Proteins for DNA replication 1. Initiator protein, 2. helicase binding to initiator protein, 3. helicase loading on DNA, 4. helicase opens the DNA and binds to primase, 5. RNA primer synthesis, 6. DNA polymerase binding and DNA synthesis

INISIASI

DENATURASI

15


Replication is bidirectional from a specific sequence of DNA called the origin of replication (OriC) DnaA (Initiator protein) binds to the 9 bp repeat sequence within OriC, causing local denaturation of the helix within the 13 bp repeat DNA helicase is recruited (by DnaA) and loaded (by DnaC), and untwists the DNA helix (use ATP) Helicase also recruits DNA primase (modified RNA pol), forming the primosome complex, which makes an RNA primer of 5 -10 nucleotides) for DNA synthesis Helical tension is relieved by DNA gyrase (topoisomerase)

Replication

PROKARYOTE



Synthesis of telomeric DNA by telomerase


Step 1 = Binding

Step 3 = Translocation

The binding-polymerization-

translocation cycle can occurs many times

This greatly lengthens one of the strands

The complementary strand is made by primase, DNA polymerase and ligase

RNA primer

Step 2 = Polymerization


II. The Eukaryotic Problem of Telomere Replication

RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

Do chromosomes get shorter with each replication???


Solution to Problem: Telomerase

• Telomerase enzyme adds TTGGGG repeats to end of lagging strand template.

• Forms hairpin turn primer with free 3’-OH end on lagging strand that polymerase can extend from; it is later removed.

• Age-dependent decline in telomere length in somatic cells, not in stem cells, cancer cells.


mtDNA replication


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HO Replikasi DNA