Structure, Replication and Recombination of DNA

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Structure, Replication and Recombination of DNA
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Information Flow From DNA
DNA
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DNA Structure
Primary Structure Chain of Nucleotides
Secondary Structure Double Helix
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DNA Structure
Nucleotide building block of DNA

• Three components of a nucleotide
• 1. Nitrogen-containing base
• purine or pyrimidine
• 2. 5-carbon sugar
• 3. Phosphate group

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DNA Structure
Purine bases Adenine (A) Guanine (G)
Pyrimidine bases Cytosine (C) Thymine (T)
5-carbon sugar Deoxyribose
Phosphate PO4
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Chemical Bonding
Covalent Bond Strong Atoms Share Electrons Formation of a Nucleotide
Hydrogen Bond Weak Atoms Share a Hydrogen Pairing of Nucleotide Bases
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Hydrogen bonds hold pairs of bases together.
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DNA Secondary Structure The Double Helix

• Two polynucleotide chains are wound together
• Bases are located inside the helix
• Sugar-phosphate groups are on the outside as a
  backbone
• Bases are arranged like rungs on a ladder,
  perpendicular to the backbone

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DNA Secondary Structure The Double Helix

• Hydrogen bonding between bases holds the chains
  together
• A pairs with T
• G pairs with C
• Polynucleotide chains have opposite polarity
• One is 5 ? 3
• other is 3 ? 5
• 10 base pairs per turn of the helix

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DNA Replication An Overview
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DNA Replication
DNA replication is semiconservative. Each strand
is used as a template to produce a new strand.
AGCTAGCTAGCT TCGATCGATCGA
TCGATCGATCGA new
AGCTAGCTAGCT new
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DNA Replication
DNA replication requires 1. DNA
polymerase, an enzyme that adds
nucleotides in a 5?3 direction. 2.
Nucleoside triphosphates 3. Energy release
of diphosphate
5A G C T 3
A 5
G
C
3 T
G
C
T 3
5 A
3T C G A5
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Origin of Replication
DNA replication begins at a replication origin
and proceeds bidirectionally, creating two
replication forks for each origin. Eukaryotic
chromosomes have multiple origins of replication.
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Continuous and Discontinuous Synthesis
DNA Polymerase builds a new strand in a 5?3
direction. This leads to continuous synthesis on
the strand oriented 3?5 and discontinuous on
the strand oriented 5?3.
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Steps in DNA Replication (Bacterial)

• Initiation
• Initiator Proteins bind to replication
  origin and cause a small section to unwind.

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Steps in DNA Replication (Bacterial)

• Unwinding
• Helicase molecules further unwind helix.
• Single-stranded binding proteins keep helix
  from reforming.DNA gyrase reduces supercoils
  ahead of replication fork.

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Steps in DNA Replication (Bacterial)

• Elongation
• Primase synthesizes a short RNA strand
  primer.
• DNA polymerase III adds nucleotides to the
  primer in a 5?3 direction.

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Steps in DNA Replication (Bacterial)

• Elongation
• A single primer is required for leading
  strand replication. On the lagging strand, a new
  primer is used at the start of each Okasaki
  fragment.

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Steps in DNA Replication (Bacterial)

• Elongation
• DNA polymerase I replaces primer RNA with
  DNA nucleotides. DNA ligase seals gaps in
  sugar-phosphate backbone.

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Steps in DNA Replication (Bacterial)

• Termination
• Termination occurs when two replication
  forks meet.
• E. coli cells have a protein called Tus
  that binds to termination sequences and blocks
  helicase movement.

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Accuracy of DNA Replication

1. Nucleotide Selection
2. DNA proofreading 3?5 exonuclease activity of
   DNA polymerase
3. Mismatch Repair repair enzymes

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Modes of Replication
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Differences for Eukaryotic DNA Replication

• Replication Licensing Factor attaches to each
  origin, initiator protein only recognizes
  licensed origins
• Multiple polymerases function in replication,
  recombination, repair
• Alpha synthesizes primer and a short stretch of
  DNA
• Delta continues replication on the lagging
  strand
• Epsilon continues replication on the leading
  strand
• Topoisomerase enzymes relax supercoils

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Replication at the Ends of Linear Chromosomes

• Removal of the primer at the end of a linear
  chromosome leaves a gap
• Linear chromosomes tend to shorten at the
  telomeres over repeated cycles of replication

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Telomerase Extends the Telomeres 3 End

• Telomerase is an enzyme composed of both protein
  and RNA
• RNA portion binds to the overhanging 3 end of
  the telomere, providing a template for elongation
• Mechanism for replicating the complementary
  strand is uncertain

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Recombination
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Holliday Model of Recombination

• Single strand breaks occur at the same
  position on homologous DNA helices.
• Single-stranded ends migrate into the
  alternate helix.

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Holliday Model of Recombination

• Each migrating strand joins to the existing
  strand, creating a Holliday junction.
• Branch point can migrate, increasing the
  amount of heteroduplex DNA.

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Holliday Model of RecombinationResolving the
Holliday Intermediate

• Separation of the duplexes requires cleavage
  in either the horizontal or vertical plane.

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Holliday Model of RecombinationResolving the
Holliday Intermediate

• Cleavage in the vertical plane, followed by
  rejoining of nucleotide strands, produces
  crossover recombinant products.

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Gene Conversion Occurs with Repair of
Heteroduplex DNA
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Gene Conversion Occurs with Repair of
Heteroduplex DNA
Gene Conversion can lead to abnormal genetic
ratios.