Rules for DNA replication

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Rules for DNA replication

• New DNA is made by coping the information from an
  existing strand of DNA (template). The new DNA
  is made in a complementary fashion to the
  template DNA.
• -Template- A structure that in some direct
  physical process can cause the patterning of a
  second structure, usually complementary to it in
  some sense.
• -Complementary base pairs- The crucial property
  of DNA is that the two strands are complementary.
• Subunits are added one nucleotide at a time. The
  incoming nucleotide added to a nascent strand is
  always a triphosphate. The reaction that ensues
  results in the liberation of a molecule of
  pyrophosphate and the nucleotide is incorporated
  into the growing chain as a monophosphate.
• The new strand is always synthesized in the 5 to
  3 direction-i.e. the first bond on the incoming
  nucleotide is at its 5 end.
• In nature DNA replication is driven by the enzyme
  DNA polymerase

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DNA replication
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DNA polymerases

• DNA polymerases are enzymes that are involved in
  the template-directed synthesis of DNA from
  dNTPs. Many enzymes qualify as DNA
  polymerases.
• DNA polymerase I was the first enzyme discovered
  with this activity.
• One of the limitations of these enzymes is that
  they cant start a new strand without a primer.
  All DNA polymerases need to be provided with this
  structure most of the time in nature an RNA
  polymerase adds the first nucleotides and the
  polymerase picks up where it left off to make a
  DNA.
• dNTPall of dTTPdGTPdCTPdATP

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• Primers- Most commonly a short nucleic acid
  sequence containing a free hydroxyl group that
  forms base pairs with a complementary template
  strand and functions as the starting point for
  addition of nucleotides to copy the template
  strand.

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Types of primers

• In the case of normal cellular DNA replication
  the primer is a short stretch of RNA that is
  synthesized on the template so that the free 3
  OH can be extended.
• A preformed RNA that can base pair with the
  template.
• A primer is generated within an existing duplex.
• A protein is bound to the DNA such that it
  provides the 3 OH.
• In vitro applications for DNA replication like
  PCR and sequencing require man made primers,
  these are always DNA. These short stretches of
  DNA are referred to as oligos.

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• DNA polymerases cannot begin a new DNA chain from
  scratch.
• Synthesis is initiation by a primosome or primase
  is required.
• The primase, which is capable of joining RNA
  nucleotides without requiring a preexisting
  strand of nucleic acid, first adds several
  comlementary RNA nucleotides opposite the DNA
  nucleotides on the parent strand. This forms what
  is called an RNA primer.

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Other functions of associated with DNA Polymerases

• There are three distinct enzymatic activities
  associated with DNA polymerase I.
• The 5-3 polymerase activity
• 5-3 exonuclease activity
• 3-5 exonuclease activity

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• The 3 to 5 exonuclease or proofreading activity
• The role of the 3'-to-5' exonuclease is to EDIT
  DNA (remove incorrectly polymerized nucleotides).
  In general, the 3'-to-5' exonuclease increases
  the accuracy or fidelity of DNA synthesis. Thus,
  when the 5' to 3' polymerization of the enzyme
  accidentally puts the wrong base into DNA, the
  3'- to 5' exonuclease proofreading activity
  immediately removes it. Errors due to
  incorporation of the wrong bases by the DNA
  polymerase are low because it must escape
  screening by two systems the base pairing rules
  recognized by the 5' to 3' catalytic site as well
  as the 3' to 5' exonuclease proofreading site.

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• 5-3 exonuclease activity
• Hydrolyses DNA from the 5 to 3 direction
  usually ahead of new DNA synthesis. This
  activity is involved in the repair of DNA and in
  the removal of RNA primers that are layed down
  during DNA replication.

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Review of DNA replication