SINTESIS DNASINTESIS DNA

ALUR INFORMASI GENETIK

Flow of information replication DNA DNAtranscription RNAtranslation protein

dsDNA

5’5’3’3’

antiparallel

dsDNA is always antiparallel

complementary

GGATGCGT

3’-CCTACGCA-5’

Two ssDNA molecules joined bystandard base-pairing rulesIn dsDNA, the strands are alwayscomplementary.

TB

5’- -3’

Bacterial DNA replicationDNA synthesis using a DNA template

Complementary base pairing (A=T, GC) determines the sequence of the newly synthesized strand.

DNA replication always proceeds from 5’ to 3’ end.

REAKSI PERPANJANGAN

Overview of bacterial DNA replication

single origin (in bacteria)bidirectionaltheta structuresreplication forksemi-conservative

TB

bacterial DNA replication

bacterialchromosome

origin (start point) bidirectional

two replication forks

thetastructure

semi-conservative

+

**

*

*

Enzim yang terlibat dalam Replikasi

SSB (ssDNA binding protein) Binds to and stabilizes ssDNA

helicaseUnwinds duplex DNA

ssDNA binding protein

binds to and stabilizes ssDNA

prevents base pairing

ssDNA binding protein

All DNA polymerases require a primer

DNA is synthesized 5' to 3'

Important facts

TB

TemplateTemplate

• A sequence of DNA or RNA that directs A sequence of DNA or RNA that directs the synthesis of a complementary the synthesis of a complementary sequencesequence

• The initial segment of a polymer that is to be The initial segment of a polymer that is to be extended on which elongation dependsextended on which elongation depends

PrimerPrimer

primasesynthesizes a short RNA primerusing a DNA template

RNA primer(a short starting sequence made of RNA)

primase

TB

PolimerasePolimerase

• Memerlukan primer dan cetakan DNAMemerlukan primer dan cetakan DNA• Polimerisasi diperpanjang pada 3’Polimerisasi diperpanjang pada 3’• Aktivitas eksonuklease Aktivitas eksonuklease 3’-5’3’-5’, berfungsi , berfungsi

sebagai proofreadingsebagai proofreading• Aktivitas eksonuklease Aktivitas eksonuklease 5’-35’-3’ untuk ’ untuk

menghilangkan primermenghilangkan primer

DNA polymerase III

Synthesizes DNA from a DNAtemplate and proofreads

TB

DNA polymerase I

Synthesizes DNA from a DNAtemplate and removes RNA primers.

DNA ligaseJoins DNA strands together by forming phosphodiester bonds

DNA ligase

replication fork

5'

5'

3'

3'

template strands

lagging strand

leading strand

helicasessDNA binding proteins

RNA primer

3'

5'Leading strand synthesis

helicase

ssDNA binding proteins

DNA polymerase

3'

5'

helicase

ssDNA binding proteins

DNA pol III

3'

5'Leading strand synthesis

DNA

helicasessDNA binding proteins

(primase)pol III

3'

Lagging strand synthesis (discontinuous) Okazaki

fragment(~1000 bases)3'

5'

Primer removalpol III

pol I

pol I

3'

5'

5’ to 3’exonucleaseactivity

Proofreading

Pol III removes misincorporated basesusing 3' to 5' exonuclease activity

This decreases the error rate to about10-10 per base pair inserted

Ligase DNALigase DNA

• Menyambung dua fragmen Okasaki Menyambung dua fragmen Okasaki dengan membentuk ikatan fosfodiester dengan membentuk ikatan fosfodiester antara 3’-OH fragmen 1 dengan 5’-P antara 3’-OH fragmen 1 dengan 5’-P fragmen 2fragmen 2

DNA ligase

Ligation

KEPERLUAN REPLIKASI DNAKEPERLUAN REPLIKASI DNA

• TEMPLATE (CETAKAN)TEMPLATE (CETAKAN)• PRIMER : 3’-OH - PERPANJANGANPRIMER : 3’-OH - PERPANJANGAN• PREKURSOR : dNTPPREKURSOR : dNTP• Enzim : polimerase DNA, helikase, Enzim : polimerase DNA, helikase,

primase, SSBP, ligaseprimase, SSBP, ligase

5

3

5

Lokus awal replikasi (Ori)Lokus awal replikasi (Ori)

INISIASI

Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic

• Bacterial cells have one giant looped Bacterial cells have one giant looped chromosomechromosome

• Replication can occur in one or two directionsReplication can occur in one or two directions– OneOne origin of replicationorigin of replication

• In Eukaryotes In Eukaryotes manymany origins of replicationorigins of replication exist exist– These form replication bubblesThese form replication bubbles– Eventually bubbles meet and replication is Eventually bubbles meet and replication is

donedone– Replication forks - where DNA is opened upReplication forks - where DNA is opened up

REPLIKON E.coliREPLIKON E.coli

REPLIKON MAMALIAREPLIKON MAMALIA

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 are 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 telomeraseHuman telomerase

• Telomerase = ribonucleoprotein complexTelomerase = ribonucleoprotein complex– Ribo = ribosomal/RNA associationRibo = ribosomal/RNA association– Nucleo = nuclear localizationNucleo = nuclear localization– Protein = contains a proteinProtein = contains a protein

• Responsible for maintaining telomere Responsible for maintaining telomere length in eukaryotic chromosomeslength in eukaryotic chromosomes

• Main components:Main components:– Telomerase reverse transcriptaseTelomerase reverse transcriptase– Human telomerase RNA (hTR)Human telomerase RNA (hTR)

Human telomerase (2)Human telomerase (2)

• Reverse transcriptaseReverse transcriptase– Transcribes RNA to DNA (rather than the Transcribes RNA to DNA (rather than the

usual DNA to RNA)usual DNA to RNA)• Telomeres – repeated regions at the end Telomeres – repeated regions at the end

of eukaryotic chromosomesof eukaryotic chromosomes• hTR is the template for the repeated hTR is the template for the repeated

regionregion

Human telomerase (3)Human telomerase (3)

• hTR 11-nt templating region consists of:hTR 11-nt templating region consists of:– Repeat template: CUAACCCRepeat template: CUAACCC– Alignment domain: UAACAlignment domain: UAAC

• Positions telomerase on the DNA strandPositions telomerase on the DNA strand• Provides template for repeat regionProvides template for repeat 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.

INHIBITOR TOPOISOMERASE

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 TARGETTOPOISOMERASE SBG TARGET

• Novobiocin – subunit ATPase GyrBNovobiocin – subunit ATPase GyrB• Asam naladiksat – Gyr AAsam naladiksat – Gyr A• Ciprofloxacin (oral) – stop replikasiCiprofloxacin (oral) – stop replikasi

MENGGANGU PROSES PEMOTONGAN DAN PENYAMBUNGAN UNTAI DNA