History

Streptococcus pneumonia galur : Experimen dg Streptococcus pneumonia galur : Smooth (S) – Virulent (gel coat) Smooth (S) – Virulent (gel coat) Rough (R) –Kurang Virulen Rough (R) – Kurang Virulen Streptococcus pneumonia galur : Experimen dg Streptococcus pneumonia galur : Smooth (S) – Virulent (gel coat) Smooth (S) – Virulent (gel coat) Rough (R) –Kurang Virulen 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 OPhosphate Group Group N Nitrogenous base (A, G, C, or T) (A, G, C, or T) CH2 O C1C1 C4C4 C3C3 C2C2 5 Sugar 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 UreaFormamid NH2  C  NH2 NH2  C  H Senyawa yang menstabilkan kondisi terdenaturasi

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 G1G1 G2G2 S phase interphase DNA replication takes place 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 troglodytes 98.77% sequence identity Identity Genetic diseases

 A.Semi-conservative B.Starts at the ‘origin’ C.Bidirectional D.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 origin Initiator proteins identify specific base sequences on DNA called sites of origin Prokaryotes – single origin site E.g E.coli - oriC Eukaryotes – 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’ direction Nucleotide recognition Enzyme catalysed polymerisation (DNA polymerase) Complementary base pair copied Substrate 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

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 II Exonuclease

Since all known DNA polymerases need a primer, how are the ends of linear 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 linear eukaryotic chromosomes Telomeres (GGGGTT) n Example n = 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' GGGGTT 5' telomerase

AACCCCAAC 5' GGGGTT primase GGGGTT

pol III pol I 5' 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