DNA replication requires unwinding of the DNA double helix by helicases. Single-stranded DNA binding proteins prevent rewinding. DNA polymerases then synthesize new strands by adding nucleotides to the 3' end of the existing strand. In eukaryotes, the leading strand is continuously extended while the lagging strand is synthesized in fragments. Proofreading by exonuclease activity increases the fidelity of replication.

1. DNA Replication and Repair

2. Unwinding and rewinding

3. Unwinding and rewinding
• Before any of these events can take place, it is
necessary for the two strands to be separated, for a
short region at least.
• This is achieved by enzymes known as helicases which
bind to the template strand and move along it,
separating the two strands.

4. • The separated strands are prevented from re-associating by
the binding of another protein, the single-stranded DNA
binding protein or SSB.
• A number of copies of the SSB will bind to the DNA strands,
maintaining a region of DNA in an extended single-stranded
form.

5. • A further complication arises from the twisting of the two
DNA strands around each other. DNA molecules within the
cell cannot normally rotate freely.

6. Topoisomerase
• In bacterial cells for example the DNA is usually circular.
• Topoisomerase
• Nick and release torsional stress

8. Fidelity of Replication: proof-reading
• It is essential that the newly synthesized DNA is a precise
(complementary) copy of the template strand.
• This does not arise simply by the nucleotides aligning
themselves in the right position, but involves the
specificity of the DNA polymerase in selecting nucleotides
that are correctly aligned.
Fidelity in this process refers to the ability of the polymerase to avoid or to
correct errors in the newly synthesized DNA strand

9. • Most DNA polymerases are more complex enzymes than
the name suggests, as they also possess exonuclease
activity.
• We have already encountered one such activity:
• the removal of the RNA primer from the Okazaki fragments is
achieved by means of the 5’ to 3’ exonuclease activity of the
DNA polymerase (i.e. it can remove bases from the 5’ end of
a chain) as it extends the following fragment.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the
end (exo) of a polynucleotide chain

10. • The fidelity of replication is enhanced by a second
exonuclease function of DNA polymerases: the 3’ to 5’
exonuclease activity, which is able to remove the
nucleotide at the growing end (3’ end) of the DNA chain.
• This is not as perverse as it sounds, since the 3’ to 5’
exonuclease only operates if there is an incorrectly paired
base at the 3’ end.

11. • The DNA polymerase will only extend the DNA chain, by
adding nucleotides to the 3’ end, if the last base at the 3’
end is correctly paired with the template strand.
• This mechanism of correcting errors, known as proof-
reading or error checking,
• adds considerably to the fidelity of replication, thus
reducing the rate of spontaneous mutation.

12. • There is a price to be paid however, as extensive error
checking will slow down the rate of replication.
• The balance between the rate of replication and the extent of
error-checking will be determined by the nature of the DNA
polymerase itself.

13. • Some DNA polymerases do not show efficient
proofreading and therefore result in a much higher
degree of spontaneous errors.
• The rate of spontaneous mutation shown by an
organism is therefore (at least in part) a genetic
characteristic that is subject to evolutionary
pressure.

14. DNA replication in
prokaryotes and eukaryotes

16. Prokaryotic DNA replication

18. The DNA Polymerase Family
A total of 5 different DNAPs have been reported in E. coli
• DNA Pol I: functions in repair and replication
• DNA Pol II: functions in DNA repair
• DNA Pol III: principal DNA replication enzyme
• DNA Pol IV: functions in DNA repair
• DNA Pol V: functions in DNA repair
To date, a total of 15 different DNA polymerases have been
reported in eukaryotes

19. Eukaryotic DNA replication

20. DNA Replication in Eukaryotes
• multiple origins of replication in eukaryotes
• human genome about 30,000 origins
• each origin produces two replication forks
• moving in opposite direction

22. DNA polymerases in eukaryotes
• Pol α : act as a primase (synthesizing an RNA primer),
elongates the primer
• Pol β : repairs DNA, (excision repair and gap-filling).
• Pol γ: Replicates and repairs mitochondrial DNA and
has proofreading 3' → 5' exonuclease activity.

23. • Pol δ: Highly possessive and has proofreading 3' →
5' exonuclease activity, responsible for replication
of lagging strand.
• Pol ε: Highly possessive and has proofreading 3' →
5' exonuclease activity, responsible for replication
of leading strand.
• η, ι, κ, Rev1 and Pol ζ are involved in the bypass of
DNA damage.
• θ, λ, φ, σ, and μ are not as well characterized
DELTA
EPSILON

24. Comparison between prokaryotic and
eukaryotic DNA replication

27. KEY CONCEPTS
DNA Replication
• Each strand in a parental duplex DNA acts as a template
for synthesis of a daughter strand and remains base
paired to the new strand, forming a daughter duplex
(semiconservative mechanism).
• New strands are formed in the 5→3 direction.

28. • Replication begins at a sequence called an origin.
• Each eukaryotic chromosomal DNA molecule contains
multiple replication origins.

29. • DNA polymerases, unlike RNA polymerases, cannot unwind the
strands of duplex DNA and cannot initiate synthesis of new
strands complementary to the template strands.
• At a replication fork, one daughter strand (the leading strand)
is elongated continuously.

30. • The other daughter strand (the lagging strand) is formed as a
series of discontinuous Okazaki fragments from primers
synthesized every few hundred nucleotides.

31. • The ribonucleotides at the 5 end of each Okazaki fragment are
removed and replaced by elongation of the 3 end of the next
Okazaki fragment.
• Finally, adjacent Okazaki fragments are joined by DNA ligase.
• Helicases use energy from ATP hydrolysis to separate the
parental (template) DNA strands.
• .

32. • Primase synthesizes a short RNA primer, which remains base-
paired to the template DNA.
• This initially is extended at the 3 end by DNA polymerase
(Pol ), resulting in a short (5)RNA- (3)DNA daughter strand

33. • Most of the DNA in eukaryotic cells is synthesized by Pol,
which takes over from Pol and continues elongation of the
daughter strand in the 5 → 3 direction.

34. • DNA replication generally occurs by a bidirectional
mechanism in which two replication forks form at an origin
and move in opposite directions, with both template strands
being copied at each fork.

35. Reference Material
• 1. Molecular Cell Biology by Harvey Lodish.
• 2. Molecular Biology of the Cell, Volume 2 by Bruce Albert
• 3. Cell and Molecular Biology by Gerald Karp