DNA Replication-III
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Initiation of Replication
• The origin of replication in E. coli is termed oriC
 origin of Chromosomal replication
• Important DNA sequences in oriC
AT-rich region
 DnaA boxes

DNA Polymerase III Is the Replicative Polymerase in E. coli
 Pol III is responsible for replicating the E. coli
chromosome.
 The Pol III core is a heterotrimer that
contains one each of α, ε, and θ subunits.
 The DNA polymerase activity is contained in
the α subunit; the γ subunit contains the
proofreading 3′→5′ exonuclease.
 The Pol III core is just one part of a much
larger protein assembly called the Pol III
holoenzyme, which replicates both leading
and lagging strands.
 The Pol III holoenzyme includes two Pol III
cores, two ring-shaped β sliding clamps, and
one clamp loader.
 The clamp loader includes two τ subunits
with C-terminal domains that protrude from
the clamp loader and bind to the Pol III cores.
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•
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The Pol III core itself is capable of DNA synthesis at a slow rate, but DNA
synthesis by the Pol III holoenzyme is exceedingly rapid, nearly 1 kb/s.
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Many Different Proteins Advance a Replication
Fork
 DNA Helicase: The two strands of the parental DNA duplex are
separated by a class of enzymes known as DNA helicases, which harness
the energy of NTP hydrolysis (usually ATP) to drive strand separation
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 Topoisomerase: As a helicase separates the parental duplex, the
strands must be untwisted.
 In E. coli, gyrase, a type II topoisomerase, is the primary replicative
topoisomerase
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 primases : synthesize short RNA primers specifically for initiating DNA
polymerase action.
 In E. coli, an RNA primer of 11 to 13 nucleotides is synthesized by the DnaG
primase.
 RNA primers are needed to initiate each of the thousands of Okazaki
fragments on the lagging strand. The leading strand is also initiated by
primase at a replication origin.
 E. coli DnaG primase must bind the DNA helicase for activity, and this
localizes its action to the replication fork
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DNA Polymerase Cannot Initiate new Strands
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 Pol I and Ligase RNA primers must be removed at the end of each
Okazaki fragment and replaced with DNA. This is achieved through
the nick translation activity of Pol I.
 The nick in the phosphodiester backbone is then sealed by DNA
ligase in a reaction that requires ATP (or NAD+ in E. coli).
 Ligase acts only on a 5′ terminus of DNA, not on RNA.
 This specificity ensures that all the RNA at the end of an Okazaki
fragment is removed before the nick is sealed.
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 SSB protein maintains the DNA template in the single strand form in order
to:
• Prevent the dsDNA formation.
• Protect the vulnerable ssDNA from nucleases.
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major elements:
•
Segments of single-stranded DNA are called template strands.
•
Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.
•
Initiator proteins and DNA helicase binds to the DNA at the replication fork and
untwist the DNA using energy derived from ATP.
•
DNA primase binds to helicase producing a complex called a primosome
•
Primase synthesizes a short RNA primer of 10-12 nucleotides.
•
Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer.
•
The RNA primer is removed and replaced with DNA by polymerase I, and the gap is
sealed with DNA ligase.
•
Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded
template DNA during the process.
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• The assembly of bacterial replication forks at the origin occurs in
steps, starting with the binding of DnaA initiator protein, which melts
an A═T-rich region.
• A DnaB helicase is then loaded onto each of the single strands of DNA
by the DnaC helicase loader.
• As DNA is unwound by DnaB, DnaG primase synthesizes RNA
primers; this is followed by entry of two Pol III holoenzymes to form
a bidirectional replication
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Termination of DNA Replication
 In E. coli, a region located halfway around the chromosome from oriC
contains two clusters of 23 bp sequences called Ter sites.
 The arrangement and orientation of Ter sites is such that bidirectional
replication forks from oriC can pass through the first set of Ter sites that they
encounter, but are blocked by the second set.
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The End Replication Problem in Eukaryotes
 At the end of a chromosome, after the leading strand has been completely extended to
the last nucleotide, the lagging strand has a single-strand DNA gap that must be primed
and filled in.
 The problem arises when the RNA primer at the extreme end is removed for
replacement with DNA .
 There is no 3′ terminus for DNA polymerase to extend from, so this single-strand gap
cannot be converted to duplex DNA.
 The genetic in formation in the gap will be lost in the next round of replication.
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The problem is solved by telomerase
 The eukaryotic cells use telomerase to maintain the integrity of DNA
telomere.
 The telomerase is composed of
Telomerase RNA
Telomerase association protein
Telomerase reverse transcriptase
 It is able to synthesize DNA using RNA as the template.
 Telomerase may play important roles is cancer cell biology and in cell
aging.
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 Telomerase carries its own template strand in
the form of a tightly bound noncoding RNA.
 Telomeres at the ends of eukaryotic
chromosomes are composed of a repeating
unit of a 6-mer DNA sequence (repeating 5′TTGGGG-3′)
 Telomerase extends the 3′ single-stranded
DNA end with dNTPs, using its internal RNA
molecule as template.
 The extended 3′ single strand of DNA is filled
in by RNA priming and DNA synthesis.
Removal of the RNA primer for this fill-in
reaction still leaves a 3′ single-stranded DNA
overhang; this end is sequestered by telomere
DNA–binding proteins.
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