TOPIC 2: DNA REPLICATION

2.1 Introduction
DNA is a biomolecule which is capable of reproducing itself in a process called replication.
Watson and Crick proposed that during replication the two DNA strands were capable of
separating and acting as templates to which a complementary set of nucleotides would attach
by base pairing to form new DNA strands.

2.2 Objectives: At the end of this unit you should be able to:
(i) Prove that DNA is capable of accurately reproducing itself.
(ii) Explain the semi-conservative mechanism of DNA replication with reference to the
experiments of Meselson and Stahl.
(iii) List the enzymes and proteins that are involved in prokaryotic DNA replication and
explain
the role of each of them.
(iv) Explain the different stages of prokaryotic DNA replication
(v) Differentiate between the synthesis of the leading strand and that of the lagging strand

2.3 Semi-conservative mechanism of DNA replication: The replication of DNA is semi-

conservative in which the original double helix molecule is duplicated into two identical
copies. Each DNA copy contains one old strand and one new strand. Evidence of this
mechanism of DNA replication came from the experiment by Meselson and Stahl who used
E.coli cells.

The cells were initially grown in a nutrient medium containing ‘heavy’ nitrogen isotope (15N)
15
for many generations until all the nitrogen in their DNA contained N. The cells were then
transferred to a medium containing the ‘light’ nitrogen isotope (14N) and different generations
of bacterial cells were sampled from the medium. The DNA in each sample was analysed by
density-gradient equilibrium centrifugation using caesium chloride (CsCl). In this method
different concentrations of CsCl were added to a centrifuge tube. The most concentrated
solution was added first followed by less concentrated solutions and the most dilute solution
was added last. This CsCl solution gradient is able to separate heavy-heavy (HH), light-light
(LL) and heavy-light (HL) DNA double helices into separate bands. The bands which were
observed in the experiment proved that DNA undergoes the semi-conservative replication
and not the conservative replication. The semi-conservative type of DNA replication has also
been conformed in eukaryotic cells
Figure 2.1.The Meselson-Stahl experiment. (a) Cells were grown for many generations in a medium containing
only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N as shown by a single band (blue) when
centrifuged in a CsCl density gradient. (b) Once the cells had been transferred to a medium containing only light
nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient
(purple band). (c) Continuation of replication for a second generation yielded two hybrid DNAs and two light
DNAs (red), confirming semi conservative replication. Adapted from Griffiths et al. 1993. An introduction to
Genetic Analysis. Page 316.

2.4 DNA replication process in prokaryotes

DNA replication in prokaryote is well represented by E.coli. The process involves the
following major steps: (i) Replication initiation, (ii) DNA denaturation – melting –unzipping,
(iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v) Separation of circular
daughter molecules, (vi) Proof reading.
2.4.1 Initiation of replication in E.coli

The replication of DNA requires the removal of the supercoils and the attachment of enzymes
and other proteins to the origin of replication on the DNA molecule. The supercoils are
removed by the enzyme called topoisomerase followed by the attachment of a protein called
DnaA. This results in the formation of the initiation complex.

Figure 2.2. Initiation of DNA replication at E.coli origin of replication.

2.4.2 Double stranded DNA denaturation

This process is also called DNA melting or unzipping. When DnaA binds to the double helix,
the first separation of the two strands takes place. This process requires ATP. This is
followed by the binding of the enzyme DNA helicase which leads to the separation of more
base pairs. The helicase moves along the DNA double helix using the energy from ATP
hydrolysis to separate the two strands. The denaturation of double stranded DNA is in the 5′
→ 3′ direction. The single strand binding protein prevents the double helix from rejoining.
Figure 2.3. DNA denaturation by the action of helicase in E.coli.

2.4.3 Formation of RNA primers in E.coli

This process is called priming. It is well known that DNA polymerase cannot initiate a new
DNA chain but can only elongate a pre-existing DNA or RNA strand. Therefore, before the
parental DNA is replicated, a short RNA molecule, complementary the DNA strand, is
synthesised by RNA polymerase. This RNA molecule is called a primer and it attaches itself
to the initiation complex. A primer is synthesised on each DNA strand in the 5′→3′ direction.

Figure 2.4. Priming on the leading and lagging strand DNA templates by the action of
primase in E.coli.
2.4.4 DNA synthesis - addition of nucleotides at the growing fork by E. coli DNA
This is a polymerization reaction catalysed by DNA polymerase. Although the two strands of
DNA double helix are anti-parallel, DNA polymerase catalyses nucleotide addition at the 3’-
hydroxyl end of each growing chain. Therefore each strand is elongated only in the 5′→3′
direction. Synthesis of a new DNA strand involves the addition of deoxyribonucleotide
triphosphates (dNTPs) which include: dATP, dGTP, dTTP, and dCTP.

2.4.4.1 Leading strand synthesis: At each growing fork, one DNA strand, called the leading
strand, is synthesized continuously from a single primer on the leading strand template. The
leading strand grows in the 5’→3’ direction, like the growing fork.

2.4.4.2 Lagging strand synthesis: Synthesis of the lagging strand is more complicated,
because DNA polymerases can add nucleotides only to the 3’ end of a primer or growing
DNA strand. Movement of the growing fork exposes the lagging-strand template on which
short RNA primers are copied. Each of these primers is then elongated by addition of dNTPs
to its 3′ end. . In E.coli, this reaction is catalysed by DNA polymerase III (poly III). The
resulting short fragments containing RNA covalently linked to DNA are called Okazaki
fragments. In bacteria and bacteriophage, Okazaki fragments contain 1000-2000 nucleotides.
The overall direction of growth of the lagging strand is from its 3′→5′ end, complementary to
the polarity of its template but opposite to the direction of nucleotide addition by DNA
polymerases. DNA polymerase I is primarily involved in removing RNA primers from
Okazaki fragments (exonuclease activity) and filling the resultant gaps using its 5′→3′
polymerizing activity. DNA ligase joins adjacent completed Okazaki fragments.

DNA polymerase II functions in the inducible SOS response and also fills gaps and appears
to facilitate DNA synthesis directed by damaged templates. DNA polymerase II resembles
DNA polymerase I in its activity, but is a DNA repair enzyme, bringing about the growth in
5′→3′ direction using free 3′- OH groups.
Figure 2.5. DNA replication at a growing fork. Source: Lodish et al. 2000. Molecular Cell
Biology, 4th edition. Page 113.

2.4.5 Proof reading by DNA polymerase

Occasionally, a wrong base is inserted during DNA synthesis. This is corrected by the
proofreading function of all bacterial polymerases.

2.4.6 Separation of circular daughter molecules

During DNA replication the parental strands remain intact and retain their superhelicity. This
poses stearic and topological constraints to the completion of the replication of a circular
DNA molecule as the two growing forks approach each other. In E.coli, decatenation of the
two daughter cells is catalyzed by Topo II (DNA gyrase) and topoisomerase IV (Topo IV).
This helps separate the daughter DNA molecules from each other
Table 2.1 Enzymes involved in prokaryotic DNA replication
# Enzyme Function
1 DnaA protein Binds DNA to form the initiation complex
2 Topoisomerase Removes coils and supercoils from DNA
3 DNA polymerase I Removes primers from the new DNA strand
4 DNA polymerase II Repairs mistakes during DNA polymerization and also fills in
the gaps left after the removal of primers from lagging strand
5 DNA polymerase III DNA polymerization
6 Helicase DNA denaturation
7 Primase Synthesis of short RNA primers on template DNA
8 Ligase Joins DNA nucleotides through covalent bonds
9 Single strand binding protein Prevents single stranded DNA from forming double strands

2.5 Revision questions: DNA replication

1. Outline the central dogma of molecular biology.

2. Explain the experiments which provided evidence in support of the mechanism of DNA
replication.
3. Differentiate between conservative and semi-conservative mechanisms of DNA
replication.
4. Describe the various stages of prokaryotic DNA replication.
5. List the enzymes and non-enzyme proteins involved in prokaryotic DNA synthesis and
explain
the role of each of them.
6. Differentiate between leading and lagging strand synthesis of prokaryotic DNA.
7. Explain how accuracy is achieved during DNA replication.

2.6 Summary: DNA replication

The DNA molecule is able to replicate itself and this is the basis of growth and reproduction among
living organisms. DNA replicates semi-conservatively with the leading strand template
replicating continuously and the lagging strand template replicating discontinuously. The
steps involved in DNA replication are (i) Replication initiation, (ii) DNA denaturation –
melting –unzipping, (iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v)
Separation of circular daughter molecules, (vi) Proof reading. All these steps are controlled
by specific enzymes.