SINTESIS DNA

Prof. Dr. Kuswandi.

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

bidirectional
origin (start point)

bacterial
chromosome
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two replication forks

theta
structure

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semi-conservative
*
*
*
+
*

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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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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)
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DNA 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'

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Leading strand
synthesis

5'

RNA primer
helicase
ssDNA binding proteins

3'

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5'

DNA polymerase
helicase
ssDNA binding proteins

3'

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Leading strand synthesis

5'

DNA pol III

helicase
DNA

ssDNA binding proteins

3'

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Lagging strand synthesis

(discontinuous)

Okazaki
fragment

3'
5'

(~1000 bases)

(primase)

helicase
ssDNA binding proteins

pol III

3'

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Primer removal
pol
III
3'
5'
pol I
pol I

5 to 3
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
10-10 per base pair inserted
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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

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 looploop interactions

Bacterial chromosomes
Plasmid antisense RNAs are generally
cis-encoded
Implies complete Watson-Crick
complementarity

Bacterial chromosomes contain transencoded 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

5'

template

RNA primer

3'

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Telomeres
repetitive DNA at the end of linear
eukaryotic chromosomes
Example
(GGGGTT)n

n = 20 - 200
GGGGTT GGGGTT GGGGTT

5'

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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

5'

GGGGTTGGGGTT

telomerase

AACCCCAAC

5'

GGGGTT GGGGTT GGGGTT

primase
5'

GGGGTT GGGGTT GGGGTT

pol III
5'
pol I
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.

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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
20 sec at 64C
1 min at 72C

Denature
Anneal
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

DNA sequence with cyclical changes in temperature

10_27_1_PCR_amplify.jpg

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

3
3

3
5

Melt template, then rapidly cool

* some primers will anneal to complementary sequence
5

5
3

3
5

Melt template, then rapidly cool

* some primers will anneal to complementary sequence
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

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
3) extend with DNA polymerase

If ever confused about how PCR works

* draw out first three cycles

25-30x

First cycle
5
3

3
5

First cycle
5
3

3
5

First cycle
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