Replikasi dan Sintesis DNA

SINTESIS DNAProf. Dr. Kuswandi.

Study objectives1. Understand how the following terms apply to DNA replication: template, complementary base pairing, origin, bi-directional, theta structures, replication fork, semi-conservative.2. Know how the following enzymes function in leading and lagging strand replication: helicase, ssDNA binding protein, primase, DNA polymerase III, DNA polymerase I. What is an Okazaki fragment?3. What is proofreading?4. Understand the problem of replicating the ends of linear DNA. Understand how telomerase solves that problem for eukaryotic chromosomes.

Flow of information replication DNA DNAtranscription RNAtranslation protein

dsDNA5533antiparalleldsDNA is always antiparallelTB

complementaryGGATGCGT3-CCTACGCA-5TB5--3

Bacterial DNA replicationDNA synthesis using a DNA templateComplementary base pairing (A=T, GC) determines the sequence of the newly synthesized strand.DNA replication always proceeds from 5 to 3 end.TB

REAKSI PERPANJANGAN

Overview of bacterial DNA replication

single origin (in bacteria)bidirectionaltheta structuresreplication forksemi-conservativeTB

bacterial DNA replicationbacterialchromosomeorigin (start point)bidirectional

two replication forksTB

semi-conservative+****TB

Key EnzymeshelicasessDNA binding proteinprimaseDNA polymerase IIIDNA polymerase IDNA ligaseTB Bacterial DNA replication

helicaseUnwinds duplex DNATB

ssDNA binding proteinbinds to and stabilizes ssDNAprevents base pairingssDNA binding proteinTB

All DNA polymerases require a primerDNA is synthesized 5' to 3'Important factsTB

TemplateA sequence of DNA or RNA that directs the synthesis of a complementary sequence

PrimerThe initial segment of a polymer that is to be extended on which elongation depends

primasesynthesizes a short RNA primerusing a DNA templateRNA primer(a short starting sequence made of RNA)primaseTB

DNA Polimerase Memerlukan primer dan cetakan DNAPolimerisasi diperpanjang pada 3Aktivitas eksonuklease 3 -5, berfungsi sebagai proofreadingAktivitas eksonuklease 5-3 untuk menghilangkan primer

DNA polymerase III

Synthesizes DNA from a DNAtemplate and proofreadsTB

DNA polymerase ISynthesizes DNA from a DNAtemplate and removes RNA primers.TB

DNA ligaseJoins DNA strands together by forming phosphodiester bondsDNA ligaseTB

replication fork5'5'3'3'template strandslagging strandleading strandTB

helicasessDNA binding proteinsRNA primer3'5'Leading strand synthesisTB

helicasessDNA binding proteinsDNA polymerase3'5'TB

helicasessDNA binding proteinsDNA pol III3'5'Leading strand synthesisTB

helicasessDNA binding proteins(primase)pol III3'Lagging strand synthesis (discontinuous)Okazaki fragment(~1000 bases)TB3'5'

Primer removalpol IIIpol Ipol I3'5'5 to 3exonucleaseactivityTB

ProofreadingPol III removes misincorporated basesusing 3' to 5' exonuclease activityThis decreases the error rate to about10-10 per base pair insertedTB

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

DNA ligaseLigationTB

KEPERLUAN REPLIKASI DNATEMPLATE (CETAKAN)PRIMER : 3-OH - PERPANJANGANPREKURSOR : dNTPEnzim : polimerase DNA, helikase, primase, SSBP, ligase

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Lokus awal replikasi (Ori)

INISIASI

oxyS/fhlA in E. colioxyS RNA transcript induced by stressfhlA transcriptional activator siteoxyS/fhlA complex binds via two loop-loop interactions

Bacterial chromosomesPlasmid antisense RNAs are generally cis-encodedImplies complete Watson-Crick complementarityBacterial chromosomes contain trans-encoded antisense RNAsNot necessarily complete complementarityOften stress-related control systems

Prokaryotic vs. EukaryoticBacterial cells have one giant looped chromosomeReplication can occur in one or two directionsOne origin of replication In Eukaryotes many origins of replication existThese form replication bubblesEventually bubbles meet and replication is doneReplication forks - where DNA is opened up

REPLIKON E.coli

REPLIKON MAMALIA

DNA triple repeats human diseaseFragile x syndrome, mental retardation, GCCHantingtons disease

INHIBITOR TOPOISOMERASEANTIBIOTIK QUINOLON MENGHAMBAT TOPOISOMERASE BAKTERI GRAM NEGATIF,MODIFIKASI BAKTERI GRAM POSITIF DAN AEROBIKCamptothecin INHIBITOR TOPOISOMERASE I SEBAGAI ANTI KANKER DENGAN MENSTABILKAN BENTUK ENZIM TERIKAT PADA DNA SECARA KOVALEN

TOPOISOMERASE SBG TARGETNovobiocin subunit ATPase GyrBAsam naladiksat Gyr ACiprofloxacin (oral) stop replikasiMENGGANGU PROSES PEMOTONGAN DAN PENYAMBUNGAN UNTAI DNA

OBAT ANTIVIRUSObat akan ikut dalam sintesis DNAStruktur pada ribosa tidak mengandung OH sintesis berhentiDiberikan dalam bentuk prodrugOleh kinase diubah menjadi trifosfat

OBAT ANTIVIRUS

Since all known DNA polymerasesneed a primer, how are the ends oflinear DNA replicated in eukaryotes?TB Replication of the ends of linear DNA

repetitive DNA at the end of lineareukaryotic chromosomesTelomeres5'TB

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.

Human telomeraseTelomerase = ribonucleoprotein complexRibo = ribosomal/RNA associationNucleo = nuclear localizationProtein = contains a proteinResponsible for maintaining telomere length in eukaryotic chromosomesMain components:Telomerase reverse transcriptaseHuman telomerase RNA (hTR)

Human telomerase (2)Reverse transcriptaseTranscribes RNA to DNA (rather than the usual DNA to RNA)Telomeres repeated regions at the end of eukaryotic chromosomeshTR is the template for the repeated region

Human telomerase (3)hTR 11-nt templating region consists of:Repeat template: CUAACCCAlignment domain: UAACPositions telomerase on the DNA strandProvides template for repeat region

AACCCCAAC5'GGGGTT5'telomerase

5'5'primase

pol IIIpol I5'ligasetelomeric repeats

For most cells, telomeres are added during development. Later telomerase becomes inactive.

Hence, as cells divide the DNA becomes shorter.TBNote that telomerase is reactivated in many types of cancer cells.

POLYMERASE CHAIN REACTION (PCR)AMPLIFIKASI FRAGMEN DNA

Example thermal cycler protocol used in lab:

Step 17 min at 94CInitial Denature

Step 245 cycles of:20 sec at 94CDenature20 sec at 64CAnneal1 min at 72CExtension

Step 37 min at 72CFinal Extension

Step 4Infinite hold at 4CStorage

BIOL 362 samples processed in: MJ Research DNA Engine Dyad

10_27_1_PCR_amplify.jpgThe polymerase chain reaction used to amplify a specific DNA sequence with cyclical changes in temperature

10_27_2_PCR_amplify.jpg

PCR applications: 1) The method of choice for amplification of relatively short DNA sequences (under 10,000 nts) can use to get genomic clone or cDNA clone

53355335Melt template, then rapidly cool* some primers will anneal to complementary sequence5353

53355335Melt template, then rapidly cool* some primers will anneal to complementary sequenceAdd DNA polymerase* provide substrate + time to extend

53355335Melt template, then rapidly cool* some primers will anneal to complementary sequenceAdd DNA polymerase* provide sunstrate + time to extend

These 3 steps constitute 1PCR cycle, which will be repeated many times (usually 25-30)1) melt template2) anneal oligonucleotide primers3) extend with DNA polymerase

If ever confused about how PCR works* draw out first three cycles25-30x

53355335First cycle

53355335First cycle

53355335First cycle

Second Cycle

Second Cycle

Second Cycle

Third cycle

Third cycle

Third cycle

From 3rd cycle onwards this species will predominateOnce it gets going truly exponential growthamplification = 2n(n = # cycles)So, 30-35 cycles, 10 billion-fold amplification- in reality, will never get this much

Third cycle

*draw cis and trans examples on board here: worth at least a couple of minutesNote that cis-acting = cis-encoded = cis-type, same for trans-*10_27_1_PCR_amplify.jpg*10_27_2_PCR_amplify.jpg