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DNA Replication MBBS

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36 views25 pages

DNA Replication MBBS

Uploaded by

godpod72
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA Replication

Dr. TUSHAR SETHI, MD


Senior Resident Biochemistry
Specific Learning
Objectives:

SLO BI 7.2.1 llustrate the processes


involved in DNA replication
Semiconservative
replication of DNA
A new copy of DNA is
synthesized with high fidelity
before each cell division
Raw material required
1) Template : Sequence information
2) Primers: 3՛–OH for new nucleotide synthesis
3) Precursor : 5՛–deoxy nucleoside triphosphate
4) Enzymes :
• DNA polymerases- dNTP polymerization
• Helicase -Processive unwinding of DNA
• Topoisomerases- Relieve torsional strain that
results from helicase-induced unwinding
• Primase- Initiates
synthesis of RNA
primers.
• DNA ligase- Seals the
single strand nick
between the nascent
chain and Okazaki
fragments on lagging
strand.
 Single-strand binding
proteins -Prevent
premature reannealing
of dsDNA strands.
DNA Polymerase Complex
• In E coli, polymerase III (pol III) functions at the
replication fork. It has highest rate of chain
elongation (1,000 dNTPs/sec), is the most
processive (can add >500,000 dNTPs before
dissociating) & accurate (error rate of 1 in 1010).
• Polymerase II (pol II) is mostly involved in
proofreading and DNA repair.
• Polymerase I (pol I) completes chain synthesis
between Okazaki fragments on the lagging strand.
• Polymerase IV: functions in DNA repair
• Polymerase V: functions in DNA repair
Eukaryotic DNA Polymerase
Eukaryotic cells have counterparts for each of these enzymes
plus some additional ones.
Replication of DNA: origins and replication
forks
Initiation of Replication

OriC
245 bp
Components of
replication fork:
• Helicase
• Primase
• DNAP
Primosome= • SSBs
Helicase+ Primase
SSB SSB

SSB SSB

ssDNA binding proteins bind to the sugar phosphate


backbone leaving the bases exposed for DNA polymerase
Supercoiling is relieved by the action of
Topoisomerases
• As the helicase continues to unwind double stranded DNA, the
segment ahead of it develops supercoiling.
• Topoisomerases form covalent intermediates with DNA in those
segments that have developed supercoiling and make nicks to
relieve the tangle.
Type I topoisomerases:
Make nicks in only
one DNA strand
Type II topoisomersases:
Make nicks in
both DNA strands
ELONGATION
DNA Polymerase: reads in 3´-5´ direction only. So, new
strand grows in 5´-3´ direction.
• Elongation of leading strand: 1 priming event
• Elongation of lagging strand: multiple priming events
DNA Synthesis by DNA polymerase III
Replication
3’
3’ 5’

5’
3’
5’ 3’ 5’ 3’5’ 3’
5’

3’
5’
3’

5’
3’
5’ 3’5’ 3’
3’5’
5’
Replication
3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Exonuclease activity of DNA polymerase I


removes RNA primers.
Replication
3’
3’

5’
3’
5’ 3’5’ 3’
5’

Polymerase activity of DNA polymerase I fills the gaps.


Ligase forms bonds between sugar-phosphate backbone.
Elongation of the leading and lagging
strands
3′→5′-Exonuclease activity enables DNA polymerase III to “proofread”
the newly synthesized DNA strand.
Excision of RNA primers and their
replacement by DNA

• DNA polymerase III continues to


synthesize DNA on the lagging
strand until it is blocked by
proximity to an RNA primer.

• When this occurs, the RNA primer


is excised and the gap filled by
DNA polymerase I

Endonuclease versus exonuclease activity


Removal of RNA primer and filling of resulting “gaps” by DNA polymerase
Formation of a phosphodiester bond by eukaryotic DNA
ligase
• Final phosphodiester linkage
between the 5′-phosphate group on
the DNA chain synthesized by DNA
polymerase III and the 3′-hydroxyl
group on the chain made by DNA
polymerase I is catalyzed by DNA
ligase

• Joining of these two stretches of


DNA requires energy, which in
eukaryotes is provided by the
cleavage of ATP to AMP + PPi
termination
• Movement of replication fork arrested with
help of a protein- terminator utilization
substance (Tus)Replication terminated.
• Detachment of daughter strands is catalyzed
by enzyme topoisomerase.
Steps involved in DNA replication in
eukaryotes:
1. Identification of the origins of replication
2. Unwinding (denaturation) of dsDNA to provide a
ssDNA template
3. Formation of the replication fork; synthesis of RNA
primer
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of the
newly synthesized DNA segments
6. Reconstitution of chromatin structure
DNA Synthesis & Replication are rigidly controlled
Difference in Eukaryotic & Prokaryotic
Process
• Process of eukaryotic DNA replication closely follows that of
prokaryotic DNA synthesis.
• There are multiple origins of replication in eukaryotic cells
versus single origins of replication in prokaryotes,.
• Eukaryotic single-stranded DNA-binding proteins and ATP-
dependent DNA helicases have been identified, whose
functions are analogous to those of the prokaryotic enzymes.
• In contrast, RNA primers are removed by RNase H and FEN1
rather than by a DNA polymerase

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