REPLIKASI DNA Dr. Oeke Yunita, S.Si., M.Si., Apt. 1
Dec 22, 2015
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
Handout Biologi Sel, Dr. Oeke Yunita
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
6 Handout Biologi Sel, Dr. Oeke Yunita
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
7 Handout Biologi Sel, Dr. Oeke Yunita
I
III III
I
DNA
8 Handout Biologi Sel, Dr. Oeke Yunita
9 Handout Biologi Sel, Dr. Oeke Yunita
Replication of Strands
Handout Biologi Sel, Dr. Oeke Yunita 10
Replication Fork
Point of Origin
Components of the DNA Replication
11 Handout Biologi Sel, Dr. Oeke Yunita
Core proteins at the replication fork
Nature (2003) vol 421,pp431-435 12 Handout Biologi Sel, Dr. Oeke Yunita
13 Handout Biologi Sel, Dr. Oeke Yunita
DNA Primer synthesis On Lagging strand
14 Handout Biologi Sel, Dr. Oeke Yunita
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
Handout Biologi Sel, Dr. Oeke Yunita
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
16 Handout Biologi Sel, Dr. Oeke Yunita
17 Handout Biologi Sel, Dr. Oeke Yunita
Synthesis of telomeric DNA by telomerase
18 Handout Biologi Sel, Dr. Oeke Yunita
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
19 Handout Biologi Sel, Dr. Oeke Yunita
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???
20 Handout Biologi Sel, Dr. Oeke Yunita
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.
21 Handout Biologi Sel, Dr. Oeke Yunita
mtDNA replication
22 Handout Biologi Sel, Dr. Oeke Yunita