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CH 14 Outline
• Chemical Nature of Nucleic Acids• Three-Dimensional Structure of DNA
– Watson and Crick• Replication
– Semi Conservative– Replication Process
• One-Gene/One-Polypeptide Hypothesis
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DNA is the Genetic Material
Therefore it must
(1) Replicate faithfully.
(2) Have the coding capacity to generate proteins and other products for all cellular functions.
• “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.”
• -Francis Crick
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The Dawn of Molecular Biology
April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
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Chemical Nature of Nucleic Acids
• DNA made up of nucleic acids– Each nucleotide is composed of a five
carbon sugar, a phosphate group, and an organic base.
nucleotides distinguished by the bases reaction between phosphate group of
one nucleotide and hydroxyl group of another is dehydration synthesis
phosphodiester bond
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Chemical Nature of Nucleic Acids
• Purines - large bases– adenine and guanine
• Pyrimidines - small bases– cytosine and thymine
Chargaff’s ruleA = T and G = C
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Three-Dimensional Structure of DNA
• X-ray diffraction suggested DNA had helical shape with a 2 nanometer diameter.
– Watson and Crick deduced DNA is an inter-twined double helix.
complementary base-pairingpurines pairing with pyrimidines
constant 2 nanometer diameter antiparallel configuration
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Models for DNA replication
1) Semiconservative model:Daughter DNA molecules contain one parental strand and one newly-replicated strand
2) Conservative model:Parent strands transfer information to an intermediate (?), then the intermediate gets copied.The parent helix is conserved, the daughterhelix is completely new
3) Dispersive model:Parent helix is broken into fragments, dispersed, copied then assembled into two new helices.New and old DNA are completely dispersed
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(a) Hypothesis 1:
Semi-conservative replication
(b) Hypothesis 2:Conservative replication
Intermediate molecule
(c) Hypothesis 3:Dispersive replication
MODELS OF DNA REPLICATION
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DNA replication Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)
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Key proposal of Watson and Crick: base pairs A : T and G : C are specific. Base pairing regulates replication.
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DNA Replication
• Since DNA replication is semiconservative, therefore the helix must be unwound.
• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.
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Replication as a process
• Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing complementary bases with theold strand.
Two molecules are made.
Each has one new and one old
DNA strand.
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Origin
5’3’
3’5’
UNIDIRECTIONAL REPLICATION
Origin
5’3’
3’5’
BIDIRECTIONAL REPLICATION
Replication can be Uni- or Bidirectional
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Semi-Conservative Replication
• Each chain in the helix is a complimentary mirror image of the other.
– double helix unzips and undergoes semi-conservative replication
each strand original duplex becomes one strand of another duplex
confirmed by Meselson-Stahl experiment
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Replication Process
• Replication of DNA begins at one or more sites (replication origin).
– DNA polymerase III and other enzymes add nucleotides to the growing complementary DNA strands.
require a primer can only synthesize in one direction
endonucleasesexonucleases
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Replication Process
• DNA polymerase cannot link the first nucleotides in a newly synthesized strand.
– RNA polymerase (primase) constructs an RNA primer.
• DNA polymerase adds nucleotides to 3’ end.– Leading strand replicates toward replication
fork.– Lagging strand elongates from replication
fork. Okazaki fragments
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Replication Process
• DNA ligase attaches fragment to lagging strand.
– Because synthesis of the leading strand is continuous and the lagging strand is discontinuous, the overall replication of DNA is referred to as semi-discontinuous.
• DNA gyrase removes torsional strain introduced by opening double helix.
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Replication Process
• Opening DNA double helix– initiating replication– unwinding duplex– stabilizing single strands– relieving torque
• Building a primer• Assembling complementary strands• Removing the primer• Joining Okazaki fragments
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Replisome
• Replisome is a macromolecular protein machine (replication organelle).
– fast, accurate replication of DNA during cell division
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Stages of Replication
• Initiation– always occurs at the same site
• Elongation– majority of replication spent in elongation
• Termination– exact details unclear