The document discusses DNA replication, including its process, key steps, and importance. It begins by outlining the learning objectives of understanding DNA replication, differentiating between prokaryotic and eukaryotic replication, determining its importance, and learning DNA repair mechanisms. The main steps of DNA replication are then described in detail: initiation involving unwinding of the DNA double helix and primer synthesis, elongation as DNA polymerase adds nucleotides to extend the DNA strands, and termination once the replication forks meet and any remaining RNA primers are removed.

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Dr Farah Deeba Khan
www.wiley.com/college/pratt/.../animations/dna_replication/index.html

4. Learning Objectives
• To understand the process of DNA replication.
• To differentiate between Prokaryotic & Eukaryotic
DNA replication.
• To determine its importance.
• To learn DNA repair mechanism.
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DNA Replication
Transcription Translation

6. Replication
The process of making an identical copy of a
section of double-stranded DNA, using
existing DNA as a template for the synthesis
of new strands.
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DNA Replication

7. Learning Outcomes
Learning Outcomes
Students should be able to answer…………..
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Learning Outcomes:
Students should be able to answer…………..

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10. Why replicate DNA?????
• If DNA never replicated,
• mitosis would slowly halve the size of the
genome until each cell would die.
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11. CELL CYCLE
Stage Activity Duration
G1 Growth & increase in cell size 10 hr
S DNA synthesis 8 hr
G2 Post-DNA synthesis 5 hr
M Mitosis 1 hr
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When DNA replicates?

12. How Replication Works????
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Models of Replication
one each of the old and new strands
Sections of the old and new pieces
duplex dispersed randomly
Old duplex is conserved with completely
new strand

14. Requirements
• DNA template
• RNA Primer (Free 3' -OH group )
• dNTP (dATP, dGTP, dCTP, dTTP)
• Proteins & Enzymes of DNA Replication
– DNA Helicases
– DNA single-stranded binding proteins (SSB-protein)
– DNA Gyrase /topoisomerase
– Primase
– DNA Polymerase
– DNA Ligase
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15. Sequential Actions
Initiation:
• Recognition of the site of start
(short sequence rich in AT base pairs)
• Unwinding & Separation of dsDNA
• Primer synthesis
Elongation:
• Add dNTPs to the existing strand
• Form phosphodiester bonds
• Correct the mismatch bases
• Extend the DNA strand
Termination:
• Stop the replication
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17. I. Initiation
1. DnaA protein binding with AT rich regions in DNA
 melting
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Initiation
replicon.

19. Initiation…..
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2. Helicases:
bind to dsDNA and stimulate separation
of the two strands.

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• Separation of dsDNA replication forks
• Two replication forks move in opposite directions
bidirectional
replicon.

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3. Single-stranded binding proteins:
bind, stabilize and keep the ssDNA
separated.
Initiation…..

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4. DNA Gyrase /Topoisomerase:
prevents formation of supercoils

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24. Topoisomerase
Type I
• negative supercoils in bacteria
• negative & positive in eukaryotes
• ATP not required
Type II
• requires ATP
• for interlocked supercoils after Chromosomal Replication
(Prokaryotes & eukaryotes)
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5. RNA Primer:
• strands of short ~ 10 bp, double stranded regions
• have RNA base pairs with a free –OH on 3’-end
• serve as starting point for DNA synthesis

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6. Primase: RNA Polymerase
adds (ATP, UTP, CTP, GTP) one at a time to 5’3’,
anti-parallel to parent DNA strand

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II. Elongation

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Direction of replication
5’3’
Elongation…

31. 7. DNA Polymerase III: adds (dATP, dTTP, dCTP,
dGTP) one at a time to RNA primer, 5’3’,
antiparallel to parent DNA strand
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Elongation….

32. DNA Polymerase Type III
• read in the 3' → 5' direction,
• adds dNTPs at rate ~800 dNTPs/S
• Pol III discriminate between correctly paired
bases and incorrectly paired bases
proofreading
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Elongation…..

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III. Termination:
•The RNA primers degraded by RNAse H &
DNA Polymerase I (exonuclease),
•gaps-filled with deoxyribonucleotides
•sealed by the enzyme ligase.

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Termination….
In prokaryotes two replication forks meet
at one point Ter

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In linear eukaryotic chromosomes,
•DNA replicated until meets another origin of replication
•Termination same

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•non-coding part of DNA at the ends of Ch….
•composed of several thousands repeats of hexameric
sequence AGGGTT
•Problem occurs at end of a linear chromosome is
reached.
•In this gap DNA is extended by telomerase.
•This extension is called a telomere

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Mechanism of Action of Telomerase

44. Functions of telomeres
• Prevent the termini from entangling & sticking
• structural integrity
• complete replication of chromosomes.
• functional organization of chromosomes within
the nucleus.
• regulation of gene expression.
• Serves as a molecular clock that controls the
replicative capacity of human cells and their
entry into senescence.
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45. • Most human somatic cells lack
telomerase activity but present
in over 90% of cancerous and
in vitro immortalized cells.
• Shorter telomeres are
associated with cellular
senescence and death.
• Diseases causing premature
aging are associated with short
telomeres.
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