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