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DNA REPLICATION
The synthesis of daughter DNA strands

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Replication Models

1) Conservative: Original molecule is conserved

2) SemiConservative: Original molecule is half conserved. It consist of

an old and a new strand.

3) Dispersive: A mixture of fragments of old and new strands

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Replication Models

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Meselson – Stahl Experiment

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Semiconservative Replication
• THE SIMPLEST MODEL
• Suggested by Watson and Crick
• Enzymes responsible:
• DNA Polymerase I, II, III etc
• DNA unwinds
• New nucleotides are added according to rules of complementarity
• A will be added to T and C to G on one strand whereas on the
other strand T will be added to A and G to C.

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5’ 3’
A T

T A

G C

C G
3’ 5’

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Replication Issues – That must be resolved
• The helix must undergo unwinding at the origins
• The open DNA helix must be stabilized so that synthesis may
proceed along both strands
• Unwinding results in increased coiling that creates tension further
down the helix, which must be reduced.
• DNA polymerase cannot begin polymerization from scratch. A
primer of must be synthesized so that polymerization can
commence. Surprisingly, RNA, not DNA, serves as the primer.
• Once the RNA primers have been synthesized, DNA polymerase III
begins to synthesize the complementary strands for the parent
molecule
• Due to antiparallel nature of DNA strands, synthesis on one template
strand is continuous but on the other strand, the process is different.
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Replication Issues – That must be resolved
• The RNA primers must be removed prior to completion of
replication.
• The gaps that are temporarily created must be filled with DNA
complementary to the template at each location
• The newly synthesized DNA strand that fills each temporary gap
must be joined to the adjacent strand of DNA.
• While DNA polymerases accurately insert complementary bases
during replication, they are not perfect, and, occasionally,
incorrect nucleotides are added to the growing strand.
• A proofreading mechanism that also corrects errors is also an
essential part of DNA synthesis.

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REPLICATION – THE PROCESS
• UNWINDING THE DNA HELIX
• Several proteins called HELICASES, bind to DNA at origin of
replication and unwind or destabilize the double helix
• Other proteins, called single-stranded binding proteins
(SSBPs), stabilize this open DNA Helix.
• As unwinding proceeds, a coiling tension is created
ahead of the replication fork, often producing supercoiling..
• Such supercoiling can be relaxed by DNA gyrase, a member of a
larger group of enzymes referred to as DNA topoisomerases.
• The gyrase makes either single- or double-stranded “cuts” and also
removes the twists and knots created during supercoiling. The
strands are then resealed. These various reactions are driven by the
energy released during ATP hydrolysis. 10
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Initiation of DNA Synthesis Using an RNA Primer
• Once a small portion of the helix is unwound, what else is needed to
initiate synthesis? As we have seen, DNA polymerase III requires a
primer with a free 3’-hydroxyl group in order to elongate a
polynucleotide chain.
• A short segment of RNA (about 10 to 12 nucleotides long),
complementary to DNA, is first synthesized on the DNA template.
• Synthesis of the RNA is directed by a form of RNA polymerase called
primase, which does not require a free 3’ end to initiate synthesis.
• It is to this short segment of RNA that DNA polymerase III begins to add
deoxyribonucleotides, initiating DNA synthesis.
• Later, the RNA primer is clipped out and replaced with DNA. This is
thought to occur under the direction of DNA polymerase I.

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Continuous and Discontinuous DNA Synthesis
• The two strands of a double helix are antiparallel to each other—that
is, one runs in the 5`–3` direction, while the other has the opposite 3`–
5` polarity.
• Because DNA polymerase III synthesizes DNA in only the 5`–3`
direction, synthesis occurs in one direction on one strand and in the
opposite direction on the other.
• As a result, as the strands unwind and the replication fork progresses
down the helix (Figure 11–11), only one strand can serve as a template
for continuous DNA synthesis.
• This newly synthesized DNA is called the leading strand.
• As the fork progresses, many points of initiation are necessary on the
opposite DNA template, resulting in discontinuous DNA synthesis of
the lagging strand.
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Okazaki Fragments
• A discontinuous form of replication takes place on the
complementary strand backward, away from the replication fork.
• It occurs in short segments, called Okazaki fragments.
• Okazaki fragments were discovered by Reiji and Tuneko Okazaki.

Replication Fork

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Continuous and Discontinuous DNA Synthesis
• Once initiated, continuous DNA replication can proceed indefinitely.
• DNA polymerase III on the leading strand template has what is
called high processivity.
• Once it attaches, it doesn't release until the entire strand is
replicated.
• Discontinuous replication, however, require the repetition of four
steps:
1. Primer synthesis,
2. Elongation,
3. Primer removal with gap filling, and
4. Ligation.
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Discontinuous DNA Synthesis on Lagging Strand
• PRIMER SYNTHESIS AND ELONGATION
• In order for Okazaki fragments to be synthesized, a primer must be created
de novo (Latin, from the beginning).
• None of the DNA polymerases can create that primer.
• Instead one of two enzymes, either
• RNA polymerase, the transcribing enzyme or, more commonly,
• Primase, an RNA polymerase coded for by the dnaG gene,
• creates the primer.
• Primer is from two to sixty nucleotides, depending on the species and it
provides the free 3'-OH group that DNA polymerase III needs in order to
synthesize the Okazaki fragment.
• DNA polymerase III continues until it reaches the primer RNA of the
previously synthesized Okazaki fragment. At that point it stops and releases
from the DNA.
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Discontinuous DNA Synthesis on Lagging Strand

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Discontinuous DNA Synthesis on Lagging Strand
• PRIMER REMOVAL AND GAP FILLING
• DNA polymerase I is a polymerase when it adds nucleotides, one
at a time, and an exonuclease when it removes nucleotides one at
a time.
• To complete the Okazaki fragment, DNA polymerase I acts in both
capacities.
• DNA polymerase I completes the Okazaki fragment by removing
the previous RNA primer and replacing it with DNA nucleotides.
• When DNA polymerase I has completed its nuclease and
polymerase activity, the two previous Okazaki fragments are
almost complete.
• All that remains is a single phospho-diester bond to be made.
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Discontinuous DNA Synthesis on Lagging Strand
• LIGATION
• DNA polymerase I cannot make the final bond to join the Okazaki
fragment to the previously synthesized DNA.
• An enzyme, DNA ligase, completes the task by making the final
phosphodiester bond in an energy-requiring reaction.

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THANK YOU

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