Dna replication notes

DNA REPLICATION
DNA replication or DNA synthesis is the process of copying
double-stranded DNA molecule.
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It is the process of copying genetic material.
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A DNA molecule is a long polymer built from nucleotides.
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Both strands in DNA run anti-parallel to each other and are
complementary to one another.
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One of the DNA strands is built in the 5′ → 3′ direction, while the
complementary strand is built in the 3′ → 5′ direction (5′ and 3’
each mark one end of a strand).
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The nucleotides which make up DNA are referred to by their
bases and when bonded are referred to as base pairs.
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Adenine pairs with Thymine and Cytosine pairs with Guanine.
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A strand running in the 5’→ 3′ direction that has Adenine will
pair with base Thymine on the complementary strand running in
3’→ 5′ direction.
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As DNA strands are antiparallel and complementary, each strand
can serve as a template for the reproduction of the opposite strand.
 The template strand is preserved in its entirety and the new strand is assembled from
nucleotide triphosphates.
 This process is called semiconservative replication.
 Ideally, the two resulting strands are identical, although in reality there are always errors,
though proofreading and error-checking mechanisms exist to ensure a very high level of
fidelity.
 In a cell, DNA replication is obligatory prior to cell division.
 Prokaryotes persistently replicate their DNA.
 In eukaryotes, timings are highly regulated and this occurs during the S phase of the cell cycle,
preceding mitosis or meiosis I.
The Replication Fork
 The replication fork is a structure which forms when DNA is replicating itself.
 It is created through the action of helicase, which breaks the hydrogen bonds holding the two
DNA strands together.
 The resulting structure has two branching “prongs”, each one made up of a single strand of
DNA.
DNA replication.
 In the first step, the double helix shown above in blue is unwound by a helicase.
 Next, a molecule of DNA polymerase III shown in green binds to one strand of the DNA.
 It moves along the strand, using it as a template for assembling a leading strand shown above
in red of nucleotides and reforming a double helix.
 A DNA polymerase I molecule (also green) is used to bind to the other template strand
(lagging strand) as the double helix opens.
 This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki
fragments).
 Another enzyme, DNA ligase shown in violet, then stitches these together into the lagging
strand.
 DNA replication begins with the “unzipping” of the parent molecule as the hydrogen bonds
between the base pairs are broken.
 Once exposed, the sequence of bases on each of the separated strands serves as a template to
guide the insertion of a complementary set of bases on the strand being synthesized.
 The new strands are assembled from deoxynucleoside triphosphates.
 Each incoming nucleotide is covalently linked to the “free” 3′ carbon atom on the pentose as
 the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
 The nucleotides are assembled in the order that complements the order of bases on the
strand serving as the template.
 Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so
on.
 When the process is complete, two DNA molecules have been formed identical to each other
and to the parent molecule.
Lagging Strand Synthesis
 In DNA replication, the lagging strand is the DNA strand at the opposite side of the replication
fork from the leading strand.
 It goes from 3′ to 5′ (these numbers indicate the position of the molecule in respect to the
carbon atoms it contains).
 When helicase unwinds DNA, two single stranded regions of DNA (the “replication fork”)
form. DNA polymerase cannot build a strand in the 3′- 5′ direction.
 Thus, the strand complementary to the 5′->3′ template strand (known as the lagging strand) is
synthesized in short segments known as Okazaki fragments.
 On the lagging strand, primase builds an RNA primer in short bursts.
 DNA polymerase is then able to use the free 3′ OH group on the RNA primer to synthesize DNA
in the 5′->3′ direction.
 The RNA fragments are then removed and new deoxyribonucleotides are added to fill the
gaps where the RNA was present.
 DNA ligase is then able to ligate the deoxyribonucleotides together, completing the synthesis
of the lagging strand.
Leading Strand Synthesis
 The leading strand is defined as the DNA strand that is synthesized in the 5′->3′ direction in a
continuous manner.
 On this strand, DNA polymerase is able to synthesize DNA using the free 3′ OH group donated
by a single RNA primer (multiple RNA primers are not used) and continuous synthesis occurs
in the direction in which the replication fork is moving.
Proteins of DNA Replication (Enzymes)
 DNA exists in the nucleus as a condensed, compact structure.
 To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the
double-stranded DNA molecule.
 These proteins are required because DNA must be single-stranded before replication can
proceed.
1. DNA Helicases
 These proteins bind to the double stranded DNA and stimulate the separation of the two
strands.
2. DNA single-stranded binding proteins
 These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that
is generated by the action of the helicases.
 Replication is 100 times faster when these proteins are attached to the single-stranded DNA.
3. DNA Gyrase
 This enzyme catalyzes the formation of negative supercoils that is thought to aid with the
unwinding process.
 In addition to these proteins, several other enzymes are involved in bacterial DNA replication.
4. DNA Polymerase
 DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is
the best characterized enzyme.
 Although this was the first enzyme to be discovered that had the required polymerase
activities, it is not the primary enzyme involved with bacterial DNA replication.
 That enzyme is DNA Polymerase III (Pol III).
 Three activities are associated with DNA polymerase I;
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5′ to 3′ elongation (polymerase activity)
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3′ to 5′ exonuclease (proof-reading activity)
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5′ to 3′ exonuclease (repair activity)
 The second two activities of DNA Pol I are important for replication, but DNA Polymerase III
(Pol III) is the enzyme that performs the 5′-3′ polymerase function.
5. Primase
 The requirement for a free 3′ hydroxyl group is fulfilled by the RNA primers that are
synthesized at the initiation sites by these enzymes.
6. DNA Ligase
 Nicks occur in the developing molecule because the RNA primer is removed and synthesis
proceeds in a discontinuous manner on the lagging strand. The final replication product does
not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3’hydroxyl and 5′-phosphate groups.
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