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REPLIKASI DNA. SINTESIS DNA. Study Objective. 1 . Understand how the following terms apply to DNA replication: template , complementary base pairing, origin, bi-directional, theta structure, replication fork, semi-conservative.

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sintesis dna

REPLIKASI DNA

SINTESIS DNA

study objective
Study Objective
  • 1. Understand how the following terms apply to DNA replication: template, complementary base pairing, origin, bi-directional, theta structure, 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. Understandhow telomerase solves that problem for eukaryotic chromosomes.
slide3

Flow of information

replication

DNA DNA

transcription 

RNA

translation 

protein

slide5

Overview of bacterial DNA replication

single origin (in bacteria)

bidirectional

theta structures

replication fork

semi-conservative

TB

slide7

bacterial DNA replication

TB

bidirectional

origin (start point)

bacterial

chromosome

slide8

theta

structure

two replication forks

TB

slide9

konservatif

semikonservatif

dispersif

dna replication
DNA Replication

Let’s meetthe team…

  • Large team of enzymes coordinates replication
keperluan replikasi dna
KEPERLUAN REPLIKASI DNA
  • TEMPLATE (CETAKAN)
  • PRIMER : 3’-OH - PERPANJANGAN
  • PREKURSOR : dNTP
  • Enzim
replication
Replication

I’d love to behelicase & unzipyour genes…

  • Unwind DNA
    • helicase enzyme
      • unwinds part of DNA helix
      • stabilized by single-stranded binding proteins

helicase

single-stranded binding proteins

replication fork

slide15

Key Enzymes

helicase

ssDNA binding protein

primase

DNA polymerase III

DNA polymerase I

DNA ligase

TB

slide16

Helicase

Unwinds duplex DNA

3'

5'

TB

slide17

ssDNA binding protein

binds to and stabilizes ssDNA

ssDNA binding protein

3'

5'

prevents base pairing

TB

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

3'

5'

slide19

Important facts

All DNA polymerases require a primer

DNA is synthesized 5' to 3'

TB

primer
Primer
  • The initial segment of a polymer that is to be extended on which elongation depends
slide21

primase

synthesizes a short RNA primer

using a DNA template

primase

3'

5'

RNA primer

(a short starting sequencemade of RNA)

polimerase
Polimerase
  • Memerlukan primer dancetakan DNA
  • Polimerisasidiperpanjangpada 3’
  • Aktivitaseksonuklease 3’ -5’, berfungsisebagaiproofreading (pembetulan pembacaan)
  • Aktivitaseksonuklease 5’-3’ untukmenghilangkan primer
slide23

DNA polymerase III

Synthesizes DNA from a DNA

template and proofreads

TB

slide25

DNA polymerase I

Synthesizes DNA from a DNA

template and removes RNA primers.

TB

slide26

DNA ligase

MenyambungduafragmenOkasaki

denganmembentukikatanfosfodiester

antara3’-OH fragmen 1 dengan 5’-P fragmen 2

DNA ligase

TB

slide27

Replicationfork

template strands

replication fork replication bubble

DNA polymerase III

3

3

3

3

3

3

3

3

3

3

3

growing

replication fork

growing

replication fork

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Replication fork / Replication bubble

leading strand

lagging strand

leading strand

lagging strand

leading strand

lagging strand

slide29

Leading strandsynthesis

3'

ssDNA binding proteins

RNA primer

helicase

5'

TB

slide30

Leading strandsynthesis

3'

ssDNA binding proteins

DNA Pol III

helicase

5'

TB

slide31

Lagging strand synthesis

(discontinuous)

3'

helicase

Okazaki

fragment

(~1000 bases)

(primase)

ssDNA binding proteins

Pol III

5'

TB

slide32

Primer removal

pol III

3'

5'

pol I

5’ to 3’

exonuclease

activity

pol I

TB

ligase dna
Ligase DNA
  • MenyambungduafragmenOkasakidenganmembentukikatanfosfodiesterantara 3’-OH fragmen 1 dengan 5’-P fragmen 2
slide34

Ligation

DNA ligase

TB

slide35

5

3

5

prokaryotic vs eukaryotic
Prokaryotic vs. Eukaryotic
  • Bacterial cells have one giant looped chromosome
  • Replication can occur in one or two directions
    • Oneorigin of replication
  • In Eukaryotes manyorigins of replication exist
    • These form replication bubbles
    • Eventually bubbles meet and replication is done
    • Replication forks - where DNA is opened up
chromosome erosion

3

3

3

3

5

5

5

5

Houston, we have a problem!

Chromosome erosion

DNA polymerase I

growing

replication fork

DNA polymerase III

RNA

Loss of bases at 5 endsin every replication

  • chromosomes get shorter with each replication
  • limit to number of cell divisions?
slide44

Replication of the ends of linear DNA

newly synthesized DNA

RNA primer

3'

5'

template

Since all known DNA polymerases

need a primer, how are the ends of

linear DNA replicated in eukaryotes?

TB

slide45

TELOMER

Telomerterletakdiujungkromosom.

Umumnyaberkode TTAGGG/AATCCC.

Mengawasiprosespembelahanselsupayatidakadarangkaian DNA yang hilang.

Telomermencegahkromosomtidaksalingmenempeldanterbelit yang bisamenyebabkanurutan gen kacaudanterjadimalfungsiselygberakibatkarsinogenikataumati.

Sebelumselmembelah, kromosomlebihdulumenggandakandirinya ( REPLIKASI), selanjutnyaberpisah.

Padareplikasi, enzim DNA polimerasemengcopydua single strand DNA denganbantuan PRIMER, suatu RNA.

SaatlahirpanjangTelomer 8.000 pasangbasa, saatusialanjuttinggal 3.000 bp.

Setiap x seltubuhmembelah, 30 s.d 200bp hilangdrtelomer.

Semakinpendektelomer, selsemakintua

telomeres

3

3

3

3

5

5

5

5

Telomeres

Repeating, non-coding sequences at the end of chromosomes = protective cap

  • limit to ~50 cell divisions

growing

replication fork

telomerase

Telomerase

  • enzyme extends telomeres
  • can add DNA bases at 5 end
  • different level of activity in different cells
    • high in stem cells & cancers -- Why?

TTAAGGG

TTAAGGG

TTAAGGG

slide47

Example

GGGGTT

GGGGTT

GGGGTT

(GGGGTT)n

n = 20 - 200

Telomeres

repetitive DNA at the end of linear

eukaryotic chromosomes

5'

TB

slide48

AACCCCAAC

telomerase

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 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
Human telomerase (2)
  • Telomeres – repeated regions at the end of eukaryotic chromosomes
  • hTR is the template for the repeated region
slide51

GGGGTT

5'

AACCCCAAC

GGGGTT

5'

telomerase

slide52

AACCCCAAC

GGGGTT

GGGGTT

GGGGTT

GGGGTT

GGGGTT

GGGGTT

5'

primase

5'

slide53

pol III

5'

pol I

ligase

telomeric repeats

slide54

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.

TB

type i topoisomerases
Type I Topoisomerases
  • One strand is “nicked,” allowing the remaining intact phosphate backbone to twist under torsion
  • The nicked strand is then rejoined
  • No net energy expenditure
type ii topoisomerases
Type II topoisomerases
  • The substrate is a point where DNA strands cross
  • One duplex strand is completely broken, and the other passed through the cut.
  • Uses energy
  • DNA gyrase is a Type II topoisomerase
slide59

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
TOPOISOMERASE SBG TARGET
  • Novobiocin – subunit ATPase GyrB
  • Asam naladiksat – Gyr A
  • Ciprofloxacin (oral) – stop replikasi

MENGGANGU PROSES PEMOTONGAN DAN PENYAMBUNGAN

UNTAI DNA

cipro ciprofloxacin
Cipro (ciprofloxacin)
  • Cipro is considered one of the most powerful antimicrobials available, though some bacteria are naturally resistant.
  • It’s the only effective treatment for inhalation anthrax
  • Fluoroquinolones (the antimicrobial class of which Cipro is a member) specifically inhibit bacterial DNA gyrase
  • Based on what you know about replication, what will happen next?
a world without dna gyrase
A world without DNA gyrase
  • How far could replication go?
obat antivirus
OBAT ANTIVIRUS
  • Obat akan ikut dalam sintesis DNA
  • Struktur pada ribosa tidak mengandung OH – sintesis berhenti
  • Diberikan dalam bentuk prodrug
  • Oleh kinase diubah menjadi trifosfat