DNA SYNTHESIS

Objectives
• Define DNA replication
• Recognize different models of DNA replication
(semiconservative, conservative and dispersive)
• Describe the replication fork
• List the components required to initiate DNA replication
• Explain the steps of DNA replication
• Differentiate leading and lagging strand
References:
Lippincott's Illustrated Reviews of Biochemistry, 5th Edition,
Unit VI, chapter29 (DNA structure, Replication and
Repair)
 Objectives
§ State the role of different enzymes used during the
DNA replication
§ Understand the various types of nucleases that
affect nucleic acid structure
§ Classify prokaryotic and eukaryotic DNA
polymerases
§ Define telomeres and telomerase express their role
in DNA stability
References:
Lippincott's Illustrated Reviews of Biochemistry, 5th
Edition, Unit VI, chapter29 (DNA structure, Replication
and Repair)
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§ DNA replication is the process by

which DNA makes a copy of itself.
§ DNA replication in prokaryotes and
eukaryotes occurs before the division of cells.
§ DNA synthesis (replication) in eukaryotes occurs
during the (S phase) of cell cycle before cell
division. DNA replication occurs once per cell
cycle. 46
=>
Miosis
23
-23

§ When the cell divides (Mitosis), each one of the

daughter cells will have a copy from these
chromosomes (DNA).
 (hormone)
by signals

3,4545
§ The major enzymatic functions carried out at the
process of DNA replication are well conserved
from prokaryotes to eukaryotes, but the
replication machinery in eukaryotic DNA
replication is a much larger complex.
§ DNA replication was proved to be
semiconservative.
 Models of DNA replication
three models of replication possible from such a scheme:
conservative, semi-conservative, and dispersive.
distribute

six Six s?. DNA" s

In conservative replication:
The two original DNA strands (old strand, known as the
parental strands) would re-basepair with each other after
being used as templates to synthesize new strands; and
the two newly-synthesized strands, known as the
daughter or complementary strands, would also
basepair with each other;
one of the two DNA molecules after replication would be
“all-old” and the other would be “all-new”.
 Models of DNA replication
In semi-conservative replication:
Each of the two parental DNA strands would act as a
template for new DNA strands to be synthesized, but after
replication, each parental DNA strand would basepair
with the complementary newly-synthesized strand just
synthesized, and both double-stranded DNAs would
include one parental or “old” strand and one daughter or
“new” strand.

In dispersive replication:
After replication both copies of the new DNAs would
somehow have alternating segments of parental DNA
and newly-synthesized DNA on each of their two strands.
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 Steps of replication
Step 1: Initiation:
A. Replication Fork Formation. Before DNA
can be replicated, the double stranded
molecule must be “unzipped” into two single
strands.
B. Primer Binding. The leading strand is the
simplest to replicate.
Step 2: Elongation.
Step 3: Termination.
INITIATION
 DnaA protein
• DnaA protein binds to specific nucleotide
sequences at the origin of replication (ori),
causing short, tandemly arranged (one after the
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• Melting is ATP-dependent, and results in

strand separation with the formation of
localized regions of ssDNA.
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 Components of replication fork
1. Proteins required for DNA strand
separation:
a. DnaA protein replications ori , I'
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b. Helicases enzyme unwinding
unzipping
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melting
c. Single-stranded DNA-binding proteins
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 DNA helicases
• These enzymes bind to ssDNA near the
replication fork, and then move into the
neighboring double stranded region, forcing
the strands apart in effect, unwinding
(unzipping) the double helix.
• Helicases require energy provided by ATP.
• Note: DnaB is the principal helicase of
replication in E. coli.
 Single-stranded DNA-binding (SSB)
proteins
• These proteins bind to the ssDNA generated by
helicases.
• The SSB proteins are not enzymes, These
proteins not only keep the two strands of DNA
separatedGin the area of the replication origin,
thus providing the single-stranded template
required by polymerases, but also protect the
DNA from nucleases that degrade ssDNA.
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 Primase
• A specific RNA polymerase, called primase (DnaG), synthesizes
the short stretches of RNA (approximately ten nucleotides long
called primers) that are complementary and antiparallel to the
DNA template. W
Mix DNA, RNA

• In the resulting hybrid duplex, the U in RNA pairs with A in DNA.

• These primers are constantly being synthesized at the replication
fork on the lagging strand (multiple primers), but only one RNA
sequence at the origin of replication is required on the leading
strand.
• The substrates for this process are 5'-ribonucleoside
2 phosphat
triphosphates, and pyrophosphate is released as each
ribonucleoside monophosphate is added through formation of a
3'→5' phosphodiester bond.
• The RNA primer is later removed.
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 DNA polymerase III
• DNA chain elongation is catalyzed by DNA polymerase III. -

• Using the 3'-hydroxyl group of the RNA primer as the acceptor of

the first deoxyribonucleotide, DNA polymerase III begins to add
nucleotides along the single-stranded template that specifies the
sequence of bases in the newly synthesized chain.
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• DNA polymerase III is a highly “processive” enzyme—that is, it

remains bound to the template strand as it moves along, and does not
diffuse away and then rebind before adding each new nucleotide.
• The processivity of DNA polymerase III is the result of its β
subunit forming a ring that encircles and moves along the template
strand of the DNA, thus serving as a sliding DNA clamp.
• The new strand grows in the 5'→3' direction, antiparallel to the
parental strand. Polymerisation
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• The nucleotide substrates are 5'-deoxy-ribonucleoside

triphosphates. Pyrophosphate (PPi) is released when each new
deoxynucleoside monophosphate is added to the growing chain.
• Hydrolysis of PPi to 2Pi means that a total of two high-energy
bonds are used to drive the addition of each deoxynucleotide.
• All four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP,
and dGTP) must be present for DNA elongation to occur.
• If one of the four is in short supply, DNA synthesis stops when
that nucleotide is depleted. ⑤s
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 Proofreading of newly synthesized
DNA
• DNA polymerase
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activity 3'→5' exonuclease.
• The proofreading exonuclease activity requires
movement in the 3'→5' direction, not 5' →3'
like the polymerase activity. This is because
the excision must be done in the reverse
direction from that of synthesis
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 DNA ligase
• The③ final phosphodiester linkage between the
5'-phosphate group on the DNA chain
synthesized by DNA polymerase III and the 3'-
hydroxyl group on the chain made by DNA
polymerase I is catalyzed by DNA ligase.
• The joining of these two stretches of DNA
requires energy, which in most organisms is
provided by the cleavage of ATP to AMP +
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TERMINATION
 Termination of replication
• Termination of DNA replication occurs when
two replication forks meet on the same stretch
of DNA.
• Any remaining gaps are filled and ligated
(sealed).
• Replication proteins are unloaded.
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 What is DNA supercoil?
• “Supercoiling” means the coiling of a coil.
• DNA is coiled in the form of a double helix, with both
strands of the DNA coiling around an axis. The further
coiling of that axis upon itself produces DNA
supercoiling.
• DNA supercoiling is generally a manifestation of
structural strain. xi
• Positive supercoiling of DNA occurs when the right-
handed, double-helical conformation of DNA is twisted
even tighter (twisted in a right-handed fashion) until the
helix begins to distort.
• Negative supercoiling, on the other hand, involves
twisting against the helical conformation (twisting in a
left-handed fashion).
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Type I DNA topoisomerases is ro' Dir & sagosts. i

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• These enzymes reversibly cut one strand of the

double helix.
• They have both nuclease (strand-cutting) and
ligase (strand-resealing) activities. release

sealing
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energy

• They do not require ATP, but rather appear to store

the energy from the phosphodiester bond they
cleave, reusing the energy to reseal the strand.
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• Each time a transient “nick” is created in one DNA

strand, the intact DNA strand is passed through the
break before it is resealed, thus relieving
(“relaxing”) accumulated supercoils. negative
• Type I topoisomerases relax supercoils in E. coli and
in eukaryotic cells. negative
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 Type II DNA topoisomerases
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• These enzymes bind tightly to the DNA double helix and make
transient breaks in both strands.
• This is an ATP-requiring process.
• Type II DNA topoisomerases are also required in both
prokaryotes and eukaryotes for the separation of interlocked
molecules of DNA following chromosomal replication.
• DNA gyrase, a Type II topoisomerase found in bacteria and
plants, has the unusual property of being able to introduce
negative supercoils into relaxed circular DNA using energy
from the hydrolysis of ATP. This facilitates the future replication
of DNA because the negative supercoils neutralize the positive
supercoils introduced during opening of the double helix.
• Some types of anticancer agents, target
human topoisomerase II.
type oftopoisomerase I
Q

• Bacterial DNA gyrase is a unique target of a

group of anti microbial agents called
quinolones, for example, ciprofloxacin.
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• Telomeres are complexes of non-coding DNA plus-

proteins located at the ends of linear chromosomes.

• They maintain the structural integrity of the
chromosome, preventing attack by nucleases.
telomeres

• In humans, it is formed of several thousand tandem

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base-paired to a complementary region of Cs and As.

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• The GT-rich strand is longer than its CA
complement, leaving ssDNA a few hundred
nucleotides in length at the 3'-end.
• The single-stranded region is thought to fold
back on itself, forming a loop structure that is
stabilized by protein.

telomeres shorten with each successive

cell division. When it is shortened beyond
some critical length, the cell is no longer able
to divide and is said to be senescent.
• It is a complex enzyme contains a protein that
acts as a reverse transcriptase, and a short piece
of RNA that acts as a template.
• The reverse transcriptase uses the RNA template
to synthesize telomere DNA.
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nucleoside analogs

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