REPLICATION OF DNA

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• REPLICATION OF DNA
• Watson and Crick were particularly excited about
  their model because the complementary nature of
  the DNA molecule suggested a way in which it
  might self-replicate. The two strands could
  separate from one another, each still containing
  the complete information, and synthesize a new
  strand. However, experiments still had to be done
  to prove that this model was true

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• DNA replication- When a new DNA strand is
  synthesized the parent DNA molecule is unwound
  and each parent strand is used as the template.
  Each replicated DNA molecule would consist of one
  old and one new strand which is called as
  semiconservative replication. In conservative
  replication the two new strands join together

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• In 1958, Mathew Meselson and Franklin Stahl
  provided strong evidence that cells use the
  semiconservative replication to produce new DNA.
• They grew E.coli cells for many generations in a
  medium where 15NH4Cl was the only N source.
  Therefore the nitrogenous bases of all DNA
  contained 15N and can be distinguished from 14N
  containing DNA by the use of sedimentation
  equilibrium centrifugation (using CsCl more
  dense DNA reaches the bottom than the lighter
  one)
• 15N cells were then transformed to a medium
  containing only 14NH4Cl, the time of transfer was
  taken as zero. E .coli cells were allowed to
  replicate over several generations, with cell
  samples removed after each replication cycle. DNA
  was isolated and subjected to sedimentation
  equilibrium centrifugation .
• Fig 11-3 and 11-4

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• The experiment provided strong support for
  semiconservative DNA replication.
• Using root tips of broad beans, Vicia faba,
  Herbert, Taylor, Woods and Hughes showed that
  semiconservative DNA replication occurs in
  eukaryotes

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• Origin of replication- The point along the
  chromosome where DNA replication is initiated.
• Replication fork- The place along the chromosome
  where the replication is initiated, the two
  strands become unwound making a replication fork.
• Starting from a point of origin, the replication
  is bidirectional creating two replication forks.
• Synthesis of DNA in microorganisms- studies on
  DNA synthesis were first reported in E coli.
• DNA Polymerase I Studies on enzymology of DNA
  replication was first reported by Arthur Kornberg
  and collegues in 1957. They isolated the enzyme
  called DNA polymerase I from E. coli that
  directed the DNA synthesis in vitro. The
  requirements needed to be satisfied for DNA
  synthesis to occur in vitro in the presence of
  DNA polymerase I are all 4 dATP, dCTP dGTP
  dTTP (dNTPs) (deoxyribonucleoside triphophates)
  and template DNA in the presence of Mg2

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• dNTPs are the precursor molecules for in vitro
  DNA synthesis. The enzyme DNA polymerase I adds
  nucleotides to an already existing DNA strand
  called a primer. These precursor dNTPs contains 3
  PO4 gps attached to the 5 carbon of d-ribose.
  Chain elongation occurs in a 3-5 direction- an
  exposed OH group at the 3 carbon and PO4 at the
  5 carbon joins together releasing 2 PO4s
• However later it was found that a more complex
  nature and a large number of enzymes were
  involved in the replication of DNA in vivo.
• The Enzymes of DNA Replication
• DNA gyrase a member of DNA Topoisomerase is
  responsible for initiation of the unwinding of
  the DNA. The tension holding the helix in its
  coiled and supercoiled structure can be broken by
  nicking a single strand of DNA.
• Helicase accomplishes unwinding of the
  original double strand, once supercoiling has
  been eliminated by the topoisomerase.

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• The two strands very much want to bind
  together because of their hydrogen bonding
  affinity for each other, so the helicase activity
  requires energy (in the form of ATP) to break the
  strands apart
• DNA polymerase proceeds along a
  single-stranded molecule of DNA, recruiting free
  dNTP's . There are different forms of DNA
  polymerase , but it is polymerase III that is
  responsible for the processive synthesis of new
  DNA strands. It needs a primer with a 3'OH group
  onto which it can attach a dNTP. DNA polymerase
  is an aggregate of several different protein
  subunits, so it is often called a holoenzyme. The
  holoenzyme also has proofreading activities, so
  that it can make sure that it inserts the right
  base, and activation of nuclease (excision of
  nucleotides) can cut away any mistakes it might
  have made.

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• Primase is actually part of an aggregate
  of proteins called the primeosome. This enzyme
  attaches a small RNA primer to the
  single-stranded DNA to act as a substitute 3'OH
  for DNA polymerase to begin synthesizing from.
  This RNA primer is eventually removed RNAase H
  and the gap is filled in by DNA ligases
• The roles of the 3 DNA polymerases (in
  prokaryotes) are
• Poly I is thought to be responsible for removing
  the primer and gap filling (RNA priming)
• Poly II is responsible for repairing DNA damaged
  by external factors ex. UV
• Poly III is the enzyme responsible for
  polymerization essential to replication.
• Both Poly I and III have 3-5 exonuclease
  activity providing proof reading function
  (exonuclease proofreading)

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• Ligase can catalyze the formation of a
  phosphodiester bond given an unattached but
  adjacent 3'OH and 5'phosphate. This can fill in
  the unattached gap left when the RNA primer is
  removed and filled in. The DNA polymerase can
  organize the bond on the 5' end of the primer,
  but ligase is needed to make the bond on the 3'
  end.
• Single-stranded binding proteins are important to
  maintain the stability of the replication fork.
  Single-stranded DNA is very labile, or unstable,
  so these proteins bind to it while it remains
  single stranded and keep it from being degraded.

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• The Steps in replication
• A portion of the double helix is unwound
  by a helicase and the replication fork progresses
  along the total length.
• The enzyme primase (derived from RNA polymerase)
  lay down a a prime sequence of few nucleotides
  complementary to the template strand for DNA
  polymerase to start replication.
• A molecule of a DNA polymerase binds to one
  strand of the DNA and begins moving along it in
  the 3' to 5' direction, using it as a template
  for assembling a leading strand of nucleotides
  and reforming a double helix.

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• Because DNA synthesis can only occur 5' to 3( in
  the new strand) , a molecule of a second type of
  DNA polymerase (epsilon, e, in eukaryotes) binds
  to the other template strand as the double helix
  opens. This molecule must synthesize
  discontinuous segments of polynucleotides (called
  Okazaki fragments). Another enzyme, DNA ligase
  then stitches these together into the lagging
  strand.

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• Proof reading and error correction during DNA
  replication are carried out by the exonuclease
  activity of DNA polymerase I and III. They detect
  and excise any mismatched nucleotides.
• Eukaryotic DNA synthesis The basic process is
  the same for the bacterial replication except for
  the complexity of the process due to the larger
  amount of DNA in eukaryotes.
• Main differences are Eukaryotic chromosomes
  have multiple replication origins in contrast to
  a single origin in bacteria. As synthesis is
  triggered at each origin site double strands open
  up at A-T rich regions. Also the rate of
  synthesis by eukaryotic DNA polymerase is much
  slower- only about 50 nucleotides/second , a rate
  about 20 times less than the comparable bacterial
  enzyme.
• In eukaryotes the DNA polymerases are much more
  complex and six different forms have been
  detected. Of the six, three (Pol a, d and e) are
  considered essential to nuclear DNA replication
  in eukaryotic cells. Two other (Pol ß and ?) are
  involved in DNA repair and one (Pol ? ) in
  mitochondrial DNA synthesis.

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• In eukaryotes, initiation of DNA synthesis is
  made by a polymerase. Two of the 4 subunits of
  the enzyme function as a primase in synthsizing
  RNA primers another subunit elongates the RNA
  primer. But this enzyme is said to have low
  processivity, (reflects the length of DNA
  synthsized by an enzyme before it dissociates
  from the template) Therefore after a short DNA
  sequence is added to the RNA primer polymerase
  switching occurs.Then Pol a dissociates and Pol d
  is replaced. This has high processivity as well
  as 3-5 exonuclease activity. This has 100 fold
  increase in the rate of synthesis compared to Pol
  a. To accomodate the increased number of
  relicons eukaryotic cells contain many more DNA
  polymerase molecules than prokaryotes

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• DNA replication and Telomeres
• Unlike the single circular chromosome of
  bacteria, eukaryotic chromosomes are linear and
  have the problem of filling the space of RNA
  primer at the end of the lagging strand. Fig
  11-16 and 11-17.
• The RNA containing enzyme telomerase adds
  telomeres (repeats of short DNA sequence
  TTAGGGG in humans) to the ends of chromosomes
  which can make a hairpin loop and facilitate the
  gap filling. The presence of telomeres preserves
  the structural integrity of chromosomes. However
  normal somatic cells contain little if any
  telomarase, and as a result telomere length
  decreases by about 100 base pairs every time a
  cells divides and acts as a clock indicating the
  cell when to stop dividing attaining senescence