REPLIKASI DNA SINTESIS DNA

ALUR INFORMASI GENETIK

Flow of information replication DNA  DNA transcription  RNA translation  protein

S.cerevisiae cell cycle

dsDNA antiparallel 5’ 3’ 3’ 5’ dsDNA is always antiparallel

Two ssDNA molecules joined by standard base-pairing rules complementary 5’- GGATGCGT -3’ 3’-CCTACGCA-5’ Two ssDNA molecules joined by standard base-pairing rules In dsDNA, the strands are always complementary. TB

Bacterial DNA replication DNA synthesis using a DNA template Complementary base pairing (A=T, GC) 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) bidirectional theta structures replication fork semi-conservative TB

bacterial DNA replication origin (start point) bidirectional bacterial chromosome

two replication forks theta structure

semi-conservative * * + *

Enzim yang terlibat dalam Replikasi SSB (ssDNA binding protein) Binds to and stabilizes ssDNA

helicase Unwinds duplex DNA

ssDNA binding protein binds to and stabilizes ssDNA prevents base pairing ssDNA binding protein

All DNA polymerases require a primer Important facts All DNA polymerases require a primer DNA is synthesized 5' to 3' TB

Template A sequence of DNA or RNA that directs the synthesis of a complementary sequence Primer The initial segment of a polymer that is to be extended on which elongation depends

synthesizes a short RNA primer using a DNA template primase synthesizes a short RNA primer using a DNA template primase RNA primer (a short starting sequence made of RNA) TB

Polimerase Memerlukan primer dan cetakan DNA Polimerisasi diperpanjang pada 3’ Aktivitas eksonuklease 3’-5’, berfungsi sebagai proofreading Aktivitas eksonuklease 5’-3’ untuk menghilangkan primer

Synthesizes DNA from a DNA template and proofreads DNA polymerase III Synthesizes DNA from a DNA template and proofreads DNA polymerase I Synthesizes DNA from a DNA template and removes RNA primers. TB

DNA ligase Joins DNA strands together by forming phosphodiester bonds DNA ligase

replication fork 5' lagging strand 3' 5' leading strand template strands 3'

Leading strand synthesis 5' RNA primer helicase ssDNA binding proteins 3'

5' DNA polymerase helicase ssDNA binding proteins 3'

Leading strand synthesis 5' DNA pol III helicase DNA ssDNA binding proteins 3'

Lagging strand synthesis (discontinuous) Okazaki fragment (~1000 bases) 3' pol III (primase) 5' helicase ssDNA binding proteins 3'

Primer removal pol III 3' 5' pol I 5’ to 3’ exonuclease activity pol I

Proofreading

Proofreading Pol III removes misincorporated bases using 3' to 5' exonuclease activity This decreases the error rate to about 10-10 per base pair inserted

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

Ligation DNA ligase

KEPERLUAN REPLIKASI DNA TEMPLATE (CETAKAN) PRIMER : 3’-OH - PERPANJANGAN PREKURSOR : dNTP Enzim : polimerase DNA, helikase, primase, SSBP, ligase

5 3 5

Lokus awal replikasi (Ori)

INISIASI

Prokaryotic vs. Eukaryotic Bacterial cells have one giant looped chromosome Replication can occur in one or two directions One origin of replication In Eukaryotes many origins of replication exist These form replication bubbles Eventually bubbles meet and replication is done Replication forks - where DNA is opened up

REPLIKON E.coli

REPLIKON MAMALIA

Replication of the ends of linear DNA 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

repetitive DNA at the end of linear eukaryotic chromosomes Telomeres repetitive DNA at the end of linear eukaryotic chromosomes (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 telomerase Telomerase = ribonucleoprotein complex Ribo = ribosomal/RNA association Nucleo = nuclear localization Protein = contains a protein Responsible for maintaining telomere length in eukaryotic chromosomes Main components: Telomerase reverse transcriptase Human telomerase RNA (hTR)

Human telomerase (2) Reverse transcriptase Transcribes RNA to DNA (rather than the usual DNA to RNA) Telomeres – repeated regions at the end of eukaryotic chromosomes hTR is the template for the repeated region

Human telomerase (3) hTR 11-nt templating region consists of: Repeat template: CUAACCC Alignment domain: UAAC Positions telomerase on the DNA strand Provides template for repeat region

5' AACCCCAAC GGGGTT GGGGTT 5' telomerase

AACCCCAAC GGGGTT GGGGTT GGGGTT 5' primase GGGGTT GGGGTT GGGGTT 5'

pol III 5' pol I ligase telomeric repeats

For most cells, telomeres are added during development 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 TARGET Novobiocin – subunit ATPase GyrB Asam naladiksat – Gyr A Ciprofloxacin (oral) – stop replikasi MENGGANGU PROSES PEMOTONGAN DAN PENYAMBUNGAN UNTAI DNA