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02 Replication Yt

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16 views51 pages

02 Replication Yt

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Wndy 0903
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DNA REPLICATION

Yudy Tjahjono, B.Sc, M.Sc. Biol.


Prokaryotic and Eukaryotic
chromosomal DNA
CHROMOSOMES
Karyotyp : Paired Chromosome
(Dovlin TM, 2002)
Chromosome
• Expression regions , Exons (genes)
• Intragenic regions, intervening regions,
Introns
• Intergenic regions
• Repeated sequence regions
• Regulatory sequences (such as: RNA-
Polymerase Gene)
Gene density
the average number of genes per Mb of genomic DNA
Gene density
The number of genes decreases as organism
complexity increases
Gene density
Factors contribute to the decreased gene density in eukaryotic
cells:
• Increases in gene size
• Increases in the DNA between genes, called intergenic
sequences.

Individual genes are longer, for 2 reasons:


• As organisms become increasingly complex, there is a significant
increase in regions of DNA required to direct and regulate
transcription, called regulatory sequences.
• Protein encoding genes in eukaryotes frequently have discontinuous
protein-coding regions (introns).
Gene density
Central Dogma
in Molecular Biology
Replication

Transcription Translation
DNA
REPLICATION

• Messelsohn-Stahl Experiments
• Stages of DNA-Synthesis
• Machinery of DNA-Synthesis
• Bidirectional Synthesis
Summary of DNA Replication
 Semi conservatively, each strand of DNA function
as a template for synthesis of its complement.
 Conducted at replicons consist of “origin of
replication” and two replication forks
(bidirectional)
 Each replication fork composed of enzyme
complex, including DNA polymerase
Also involve primase, helicase, topoisomerase
DNA-REPLICATION
Three possible mechanism

? ?
DNA-REPLICATION
Messelsohn-Stahl Experiments

First generation

MIXTURE
Second
Of Bactery with labeled medium
generation
14N 15N
DNA-REPLICATION
Messelsohn-Stahl Experiments
DNA-REPLICATION
Messelsohn-Stahl Experiments
DNA-REPLICATION
is semi conservative
DNA-REPLICATION
is semi conservative
• Each strand of DNA function
as a template for synthesis
of new complement DNA,
yields 2 DNA molecules.

• Need separation of DNA


strands (part melting, or
denaturation), so that the
older strand become the
template.
STAGES OF DNA-REPLICATION
Denaturation of DNA
Component of DNA-Synthesis
• Primer
• Template
• Substrate : Desoxynucleoside Triphospate
• enzymes
Review:
Purine and Pyrimidine Base

Watson-Crick base pairing


2 key substrates:

Deoxynucleoside
triphosphates:
dGTP, dATP, dCTP, dTTP

a primer :
template junction
DNA-SYNTHESIS
Desoxyribonucleotid
Triphosphate
Primer

Template

G = -7 kcal/mole; Keq = 105


DNA-SYNTHESIS
The growth of DNA strand
The growth of
5’ 3’
replication fork

3’ 5’
The growth of DNA strand
THE MACHINERY OF DNA REPLICATION
DNA-Pol. III
DNA-Pol. III
LEADING AND LAGGING STRAND
• Made because synthesis of DNA
happen only in the direction of
3‘
5’ to 3’.
leading

5‘ • Both DNA strands experience a


3‘
different process:
– DNA synthesis on ‘leading’ strand
lagging
happen ‘continuously’
– DNA synthesis on ‘lagging’ strand
5‘
happen ‘discontinuously’
• Form an Okazaki fragment
• Okazaki fragments will be linked
together by DNA ligase activity.
OKAZAKI FRAGMENT
THE REPLICATION FORK
Synthesis of DNA „Lagging Strand“
There should be a
primer (i.e. 3’-OH) to
start the synthesis of
DNA

Primase function to
RNAse-H and make an RNA primer
The DNA fragments
should be linked
together.

This process will be


done by DNA ligase
Filling the „Gap“
DNA LIGASE

LIGASE
TOPOISOMERASE
➔Act as a ‘swivelase’.
➔Remove supercoils
produced by DNA
unwinding at the
replication fork.
➔By breaking either one
or both strands of DNA
without letting go of the
DNA and passing the
same number of DNA
strands through the
break.
HELICASE
➔ Catalyze the separation of
the two strands of duplex
DNA, at the replication fork.
➔ Bind to and move
directionally along ssDNA
using the energy of ATP
hydrolysis to displace any
DNA strand that is annealed
to the bound ssDNA.
➔ Hexameric proteins that
assume the shape of a ring
➔ Can have a polarity of either
5’→3’ or 3’→5’.
DNA-Polymerase I

DNA-Repair Machinery
Bidirectional DNA-Synthesis
PRIMASE
➔ A special RNA polymerase
➔ Dedicated to making short, RNA primers (5-10 nucleotides
long) on an ssDNA template.
➔ The primers are subsequently extended by DNA
polymerase.
➔ Does not require specific DNA sequences to initiate
synthesis of a new RNA primer.
➔ Is activated only when it associated with other DNA
replication protein, such as DNA helicase.
➔ Associated with RNAse-H and DNA-Polymerase I that
remove RNA primers to complete DNA replication, and
DNA ligase to repair the ‘nick’ formed, by creating a
phosphodiester bond between an adjacent 5’ phosphate
and 3’ OH.
Single Strand Binding Proteins
➔ Proteins that bind to the separated DNA strand
rapidly after being unwind by DNA helicase
➔ To stabilize the separated strands as template for
DNA synthesis.
➔ Cooperative binding: binding of one SSB promotes
the binding of another SSB to the immediately
adjacent ssDNA.
ORI
Origin of Replication
• DNA replication starts from a specific
nucleotide sequences, called as ‘origins of
replication’ ➔ stretch of AT-rich DNA
– Known by a protein that function to open the DNA
strand twist ➔ initiator protein.
• DNA synthesis is done at two direction from
the ‘origin’
DNA-Replication is Bidirectional
DNA synthesis occurs in replicons
consisting of an origin of replication and
two diverging replication forks
(bidirectional)

Fork movement Fork movement

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