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SINTESIS DNA

Prof. Dr. Sudjadi, Apt., MS.


Study objectives
1. 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 DNA
transcription
RNA
translation
protein
dsDNA
antiparallel

5 3
3 5

dsDNA is always antiparallel


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complementary
5- GGATGCGT -3

3-CCTACGCA-5
Two ssDNA molecules joined by
standard base-pairing rules
In dsDNA, the strands are always
complementary.
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Bacterial DNA replication
DNA 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.
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REAKSI PERPANJANGAN
Overview of bacterial DNA replication

single origin (in bacteria)


bidirectional
theta structures
replication fork
semi-conservative

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bacterial DNA replication
origin (start point) bidirectional

bacterial
chromosome

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two replication forks

theta
structure

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semi-conservative

*
*

*
+
* TB
Bacterial DNA replication
Key Enzymes
helicase
ssDNA binding protein
primase
DNA polymerase III
DNA polymerase I
DNA ligase
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helicase
Unwinds duplex DNA

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SUPERKOIL-TOPOISOMERASE
ssDNA binding protein
binds to and stabilizes ssDNA

prevents base pairing

ssDNA binding protein


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Important facts
All DNA polymerases require a primer

DNA is synthesized 5' to 3'

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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
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
DNA polymerase III
Synthesizes DNA from a DNA
template and proofreads

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DNA polymerase I

Synthesizes DNA from a DNA


template and removes RNA primers.

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DNA ligase
Joins DNA strands together by
forming phosphodiester bonds

DNA ligase

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replication fork
5'
lagging strand
3'

5'
leading strand

template strands
3' TB
Leading strand 5'
synthesis

RNA primer

helicase
ssDNA binding proteins
3' TB
5'

DNA polymerase

helicase
ssDNA binding proteins
3' TB
Leading strand synthesis 5'

DNA pol III

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

5'
pol I

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

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

DNA ligase

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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
oxyS/fhlA in E. coli
oxyS RNA transcript
induced by stress
fhlA transcriptional
activator site
oxyS/fhlA complex
binds via two loop-loop
interactions
Bacterial chromosomes
Plasmid antisense RNAs are generally cis-
encoded
Implies complete Watson-Crick
complementarity
Bacterial chromosomes contain trans-
encoded antisense RNAs
Not necessarily complete complementarity
Often stress-related control systems
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
DNA triple repeats human disease

Fragile x syndrome, mental retardation,


GCC
Hantingtons disease
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
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
OBAT ANTIVIRUS
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?

newly synthesized DNA


RNA primer

5' 3'
template TB
Telomeres
repetitive DNA at the end of linear
eukaryotic chromosomes
Example
(GGGGTT)n n = 20 - 200

GGGGTT GGGGTT GGGGTT


5' TB
Telomerases are enzymes that add
DNA repeats to the 3' end of DNA.

Telomerases are
composed of protein and
an RNA molecule that AACCCCAAC

functions as the template


telomerase
for telomere synthesis.
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
GGGGTTGGGGTT
5'
telomerase
AACCCCAAC
GGGGTT GGGGTT GGGGTT
5'

primase

GGGGTT GGGGTT GGGGTT


5'
pol III

5'
pol I

ligase

telomeric repeats
Telomerase Demo created by Dr. Donald F. Slish at SUNY, Plattsburgh.

Note -on the telomerase enzyme, T's are used in the RNA template. This was an inadvertent
error. Since this template is RNA, these should be U's

If you have questions or comments send me a message at slishdf@splava.cc.plattsburgh.edu


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.
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POLYMERASE CHAIN
REACTION (PCR)
AMPLIFIKASI FRAGMEN DNA
Example thermal cycler protocol used in lab:

Step 1 7 min at 94C Initial Denature

Step 2 45 cycles of:

20 sec at 94C Denature


20 sec at 64C Anneal
1 min at 72C Extension

Step 3 7 min at 72C Final Extension

Step 4 Infinite hold at 4C Storage

BIOL 362 samples processed in:


MJ Research DNA Engine Dyad
The polymerase chain reaction used to amplify a specific
10_27_1_PCR_amplify.jpg
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
5 3
5 3
3 5
3 5

Melt template, then rapidly cool


* some primers will anneal to complementary sequence
5 3

3 5
5 3
3 5

Melt template, then rapidly cool


* some primers will anneal to complementary sequence
5 3

3 5

Add DNA polymerase


* provide substrate + time to extend
5 3
3 5

Melt template, then rapidly cool


* some primers will anneal to complementary sequence
5 3

3 5

Add DNA polymerase


* provide sunstrate + time to extend
These 3 steps constitute 1PCR cycle, which will be repeated
many times (usually 25-30)
1) melt template
2) anneal oligonucleotide primers 25-30x
3) extend with DNA polymerase

If ever confused about how PCR works


* draw out first three cycles
First cycle

5 3
3 5

5 3

3 5
First cycle

5 3
3 5

5 3

3 5
First cycle

5 3
3 5

5 3

3 5
Second Cycle
Second Cycle
Second Cycle
Third cycle
Third cycle
Third cycle
From 3rd cycle onwards this species will predominate

Once it gets going truly exponential growth


amplification = 2n
(n = # cycles)
So, 30-35 cycles, 10 billion-fold amplification
- in reality, will never get this much
Third cycle