DNA Replication-III

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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

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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 a, e, and ? subunits.
• The DNA polymerase activity is contained in the
  a subunit the ? 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 ß sliding clamps, and one
  clamp loader.
• The clamp loader includes two t subunits with
  C-terminal domains that protrude from the clamp
  loader and bind to the Pol III cores.

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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 (gt200)
  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 DNAbinding proteins.