Very Concise note on DNA REPLICATION for 1st year Medical Students

1. DNA REPLICATION
Daniel Onyejesi

2. INTRODUCTION
• In DNA replication, the DNA copies itself
and the genome is copied.
• For the sake of simplicity in learning, the
simple organism, E.coli will be used as an
example and it has only one point where
replication can start while man has several
points where replication can start.

3. Enzymes Involved
DNA polymerases
Helicase enzyme (Helicases)
Topoisomerases

4. DNA polymerases
• The key enzymes are the DNA
polymerases (both in eukaryotes and
prokaryotes).
• In E.coli, there are 5 DNA polymerases
written as:
DNA polymerase I
DNA polymerase II
DNA polymerase III
DNA polymerase IV
DNA polymerase V

5. DNA polymerases contd
 DNA polymerase I is used in replication
• It can also remove primer molecule
• It has editing (proof-reading) function and can
remove mismatched nucleotide in 5՚-3՚ direction and
3՚-5՚ direction and replace it with the right
nucleotide
 DNA polymerase II: Can only repair
 DNA polymerase III: Essentially involved in
replication and very fast in replication. It is
used/seen in man
 DNA polymerase IV and V are only involved in
repair.
 DNA polymerase III is more sophisticated and
complicated in function than DNA polymerase I

6. CHARACTERISTICS OF DNA
POLYMERASES
They catalyze step by step addition of
deoxynucleotide into DNA chain
[DNA]n + dNTP [DNA]n+1 + PPi
For DNA polymerase to be active, all the
four nucleotides must be present (i.e.
dCTP, dTTP, dATP, dGTP,) and Mg2+
DNA polymerase normally requires a
template (i.e. a pre-existing DNA) which
determines the sequence.

7. CHARACTERITICS OF DNA
POLYMERASES contd
DNA polymerase requires a primer which
is a shoet length of RNA nucleotide which
binds to a sequence to which it is
complementary to. The primer has a 3՚
end which has a free OH group that DNA
polymerase will use to add the first
nucleotide.
A mistake can be repaired by DNA
polymerase’s ability to repair i.e. They can
proof-read synthesized strands

8. Other notes…
• DNA polymerase has an exonuclease
activity which enables it remove wrongly
attached nucleotides
• In humans DNA polymerase III can
incorporate about 3 billion nucleotides in
few seconds
• The wrong nucleotides are removed
before DNA polymerase III can start
functioning

9. HELICASE ENZYME
• These separate the double strands of the
DNA helical structure, using energy in
form of ATP.

10. TOPOISOMERASES
• DNA is normally negatively supercoiled
• Topoisomerase I relaxes the supercoil and
topoisomerase II re-creates the supercoil
after replication, this uses ATP
• In bacteria, topoisomerase II is called
DNA gyrase

11. PROCESS OF REPLICATION
• Chromosome of E.coli is circular unlike
humans that have linear chromosome
• Replication in E.coli begins at a single site
while humans have up to 3,000 sites where
replication starts at the same time.
• The site where replication [in E.coli] starts is
a unique one and is called ORIGIN OF
REPLICATION or “Ori C locus”.
• It has 245 base pairs and contains a unique
four (4) repeats of a sequence which act
together as initiation site where the initiation
protein binds i.e. Dna A.

12. PROCESS OF REPLICATION contd
• Replication starts with initiation by the binding
of the Dna A. This binding of DnaA to Ori C locus
is the beginning of the replication process.
• After Dna A bonds, other proteins like DnaB
binds to the same site.
• DnaB is the helicase enzyme which uses ATP to
unwind the double stranded DNA
• If after inwinding nothing happens, reannealing
of the 2 strands occurs
• The protein SSB (Single strand binding protein)
binds to the newly formed single strand and
prevents reannealing.

13. PROCESS OF REPLICATION contd
• This entire unit above is called pre-priming
complex, which is made up of:
DNA template
Dna A
Dna B (Helicase)
SSB protein
• After the formation of the prepriming
complex, a primase enzyme synthesizes RNA
primer which binds to the single stranded
DNA template
• This forms a multi subunit called primosome
[primosome = prepriming complex + primer]

14. PROCESS OF REPLICATION contd
• Primase is also called RNA polymerase and
it synthesizes a primer which has a short
length of about 10 nucleotides.
5՚
3՚
3՚
5՚
A replication fork

15. PROBLEM OF ANTIPARALLEL
STRANDS
• All DNA polymerases synthesize only in 5՚-3՚ direction
• At the open 3՚ end, a primer is formed in a 5՚-3՚ direction
and this strand formation move along the same direction as
helicase and is called the leading strand which is also
synthesized continuously.
• At the open 5՚ end, the strand formed does not attach to
the 5՚ end but formation starts from somewhere within, so
that the strand can be formed in a 5՚-3՚ direction
• This strand is formed away from the direction of progress
of helicase and is produced in discontinuous
strands/fragments called okazaki fragments
• This strand is called the lagging strand and the
discontinuous strands are held together by the enzyme
“ligase”.
• NB: The lagging strand is synthesized in fragments called
okazaki fragments.

16. PRACTICE QUESTIONS
• Give a brief account of DNA replication in
procaryotes
• Classify and characterize the different
types of DNA polymerases
• How is the problem of antiparallel strands
solved during DNA replication

17. References:
• Prof P.O.J Ogbunude notes on Nucleic
Acids
• Lippincott Illustrated reviews:
Biochemistry
• Lehninger principles of Biochemistry