Post on 23-Feb-2016
description
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DNA DAN REPLIKASI
History
Experimen dg Streptococcus pneumonia galur : Smooth (S) – Virulent (gel coat)
Rough (R) – Kurang Virulen
R strain could become virulent when it took in DNA from heat-killed S strain
Structure
DNA Nucleotide
O=P-O O
Phosphate Group
NNitrogenous base (A, G, C, or T)
CH2
O
C1C4
C3 C2
5
Sugar(deoxyribose)
O
Deoksi adenosin monofosfat
Deoksi guanosin monofosfat
Deoksi timidin monofosfat
Deoksi sitidin monofosfat
Melting and Renaturation of DNA
Renaturation driven by homologous base pairing
Will also hybridize with a radiolabeled 5’-ACGGCTA-3’ “probe”.
O O
Urea Formamid
NH2 C NH2 NH2 C H
Senyawa yang menstabilkan kondisi terdenaturasi
Replication
Replication
Process of duplication of the entire genome prior to cell division
In eukaryotes , replication only occurs during the S phase of the cell cycle.
Synthesis Phase (S phase)• S phase during interphase of
the cell cycle• Nucleus of eukaryotes
Mitosis-prophase-metaphase-anaphase-telophase
G1 G2
Sphase
interphase
DNA replication takesplace in the S phase.
DNA replication occurs with great fidelity(New cells will need identical DNA strands))
Somatic cell DNA stability and reproductive-cell DNA stability are essential. Why?
Pan troglodytes98.77% sequence identity
Identity
Genetic diseases
A. Semi-conservativeB. Starts at the ‘origin’C. BidirectionalD. Semi-discontinuous E. Synthesis always in the 5-3’ direction F. RNA primers required
Basic rules of replication
DNA replication
Of the 3 possible models,
replication is…
A) Semi-conservative
Meselson-Stahl
experiments
B) Starts at originInitiator proteins identify specific base
sequences on DNA called sites of origin
Prokaryotes – single origin site E.g E.coli - oriCEukaryotes – multiple sites of origin (replicator)E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes Eukaryotes
Bidirectional replication of circular DNA molecules.
Temporal ordering of DNA replication initiation events in replication units of eukaryotic chromosomes.
C) bidirectional
Replication forks move in one or opposite directions
Anti parallel strands replicated simultaneously Leading strand synthesis continuously in 5’– 3’ Lagging strand synthesis in fragments in 5’-3’
D) Semi-discontinuous replication
E) Synthesis is ALWAYS in the 5’-3’ directionNucleotide recognitionEnzyme catalysed polymerisation (DNA polymerase)Complementary base pair copiedSubstrate used is dNTP
F) RNA primers required• DNA polymerase can only join an incoming nucleotide to one that
is base-paired
• RNA primase provides a base paired 3’ end as a starting point for DNA pol by synthesising ~10 nucleotide primers
Animasi replikasi
Enzim dalam Replikasi DNA
SSB (ssDNA binding protein) Binds to and stabilizes ssDNA
exonuclease 3’-5’
exonuclease 5’-3’
Where does energy for addition of nucleotide come from?
What happens if a base mismatch occurs?
DNA polymerase has 3’ 5’ exonuclease activity in order to correct errors
From cleavage of high energy phosphate of incoming triphosphate
Why does DNA replication only occur in the 5’ to 3’ direction?
DNase I
DNase IIExonuclease
Since all known DNA polymerasesneed a primer, how are the ends oflinear DNA replicated in eukaryotes?
5' 3'
RNA primer
template
newly synthesized DNA
Replication of the ends of linear DNA
repetitive DNA at the end of lineareukaryotic chromosomes
Telomeres
(GGGGTT)n
Example
n = 20 - 200
GGGGTT GGGGTT GGGGTT
5'
Telomerases : enzymes that add DNA repeats to the 3' end of DNA.
Telomerases are composed of protein and an RNA molecule that functions as the template for telomere synthesis.
AACCCCAAC
telomerase
Human telomerase
Responsible for maintaining telomere length in eukaryotic chromosomes
Main components: Telomerase reverse transcriptase Human telomerase RNA (hTR)
Reverse transcriptase Transcribes RNA to DNA (rather than the
usual DNA to RNA) hTR is the template for the repeated
region
AACCCCAAC
5'GGGGTTGGGGTT
5'
telomerase
AACCCCAAC
5'
5'GGGGTTGGGGTT GGGGTT
primase
GGGGTT GGGGTT GGGGTT
pol III
pol I5'
ligase
telomeric repeats
For most cells, telomeres are added during development. Later telomerase becomes inactive.
Hence, as cells divide the DNA becomes shorter.
Note that telomerase is reactivated in many types of cancer cells.
OBAT anti REPLIKASI DNA
INHIBITOR TOPOISOMERASE (Gyrase)
Antibiotik QUINOLON : MENGHAMBAT TOPOISOMERASE BAKTERI GRAM NEGATIF,MODIFIKASI BAKTERI GRAM POSITIF DAN AEROBIK
Camptothecin : INHIBITOR TOPOISOMERASE I SEBAGAI ANTI KANKER DENGAN MENSTABILKAN BENTUK ENZIM TERIKAT PADA DNA SECARA KOVALEN
TOPOISOMERASE SBG TARGET OBAT
Novobiocin – subunit ATPase GyrB Asam naladiksat – Gyr A Ciprofloxacin (oral) – stop replikasi
MENGGANGU PROSES PEMOTONGAN DAN PENYAMBUNGAN UNTAI DNA