The Central Dogma of Molecular Biology

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The Central Dogma of Molecular Biology
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Lecture 1 DNA

• What is it?
• How is it organized?
• What is its structure?
• How is it replicated?
• Terminology (structure) Chromosome, gene,
  nucleosome, histone, nucleotide, sugar-phosphate
  backbone, 5 end, 3 end
• Terminology (synthesis) DNA polymerase,
  helicase, primase, leading strand, lagging
  strand, Okazaki fragment, RNA primer

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Background Reading Molecular Biology of the
Cell, 4th Edition Alberts, Johnson, Lewis, Raff,
Roberts and Walter Chapter 4 DNA and
Chromosomes pp191-208, DNA structure pp
208-216, histones Chapter 5 DNA Replication,
Repair and Recombination pp238-250, DNA
replication pp267-275, DNA repair pp275-278,
recombination pp 286-289, transposons
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Chromosomes
GENES
DNA
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Entire set of human chromosomes Karyotype
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• DNA is replicated and genes are expressed during
  interphase
• Mitosis nuclear division
• Cell division two daughter cells each with
  one copy of each chromosome

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The Functional Anatomy of a Chromosome
Telomere
Replication origin
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DNA is packed into highly condensed structures
called chromatin.
Several levels of chromatin packing gives rise to
the highly condensed mitotic chromosome.
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The Structure of DNA
James Watson, Francis Crick and Maurice Wilkins
were awarded the Nobel Prize in 1962 for their
discovery of the structure of DNA.
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• Nucleotides are the builiding blocks of DNA
  Nitrogenous base sugar phosphate
• Nitrogenous base adenine, guanine, cytosine,
  thymine
• Sugar deoxyribose

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A single strand of DNA consists of bases attached
to a sugar-phosphate backbone. Bases Guanine,
Adenine, Thymine, Cytosine DNA has POLARITY 5
end, 5 phosphate 3 end, 3 OH of sugar
BUTDNA is double-stranded!!
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5
3

• The two strands are arranged antiparallel to
  each other
• The two strands are held together through
  hydrogen bonds between the bases
• Bases on the inside of the helix,
  sugar-phosphate backbone on the outside
• Each strand contains a sequence of nucleotides
  that is complementary to the other strand

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5
5
3
3
5
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Complementary base pairs in the DNA double helix.
Single ring base pyrimidine T, C Double ring
base purine A, G Rule of base pairing
A-T G-C ALWAYS!!!!!
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The structure of the DNA double helix
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Implications of the discovery of the structure of
DNA

• Provides the molecular mechanism of how DNA is
  packaged into chromosomes

• Provides the molecular mechanism of how DNA is
  copied (replicated)

• Chemical basis of heredity provides an
  explanation for the CENTRAL DOGMA How DNA
  provides the instructions to produce proteins.

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DNA is packaged into highly ordered
structures Chromatin complex of proteins and
nucleic acid Proteins Histones
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The Nucleosome is the Basic Unit of Chromosomal
Organization
Beads on a string model of nucleosome
arrangement
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• Each individual nucleosome core consists of a
  histone octamer 146 bp DNA

• Histone octamer 2 molecules each of 4 histone
  proteins

• The double helix is wrapped around the histone
  in areas of maximum flexibility

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Chromatin is tightly packed.but there has to be
some flexibility, because DNA has to be
replicated DNA has to be transcribed into RNA for
gene expression
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The structure of the nucleosome can be altered by

• Chromatin remodelling histoneDNA contact is
  loosened/positioned differently

Histone modification by enzymes in the nucleus
ALL of these changes are Reversible.
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DNA is comprised of two complementary strands of
nucleotides in a double helix configuration. DNA
is wound around histones to form
nucleosomes. Nucleosomes are the basic structure
of chromatin Chromatin is condensed into
chromosomes
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Implications of the discovery of the structure of
DNA

• Provides the molecular mechanism of how DNA is
  packaged into chromosomes

• Provides the molecular mechanism of how DNA is
  copied (replicated)

• Chemical basis of heredity provides an
  explanation for the CENTRAL DOGMA How DNA
  provides the instructions to produce proteins.

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Lecture 1 DNA

• What is it?
• How is it organized?
• What is its structure?
• How is it replicated?
• Terminology (structure) Chromosome, gene,
  nucleosome, histone, nucleotide, sugar-phosphate
  backbone, 5 end, 3 end
• Terminology (synthesis) DNA polymerase,
  helicase, primase, leading strand, lagging
  strand, Okazaki fragment, RNA primer

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Each strand of DNA serves as a Template for
replication
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The nature of DNA replication is
Semiconservative Each cycle of replication
results in a doubling of the number of DNA
molecules ( strands molecules)
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The replication of DNA involves many enzymes
DNA polymerase - addition of nucleotides to the
new strand DNA helicase - unwinds the double
helix to expose the two strands DNA primase -
lays down RNA primers for lagging strand synthesis
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Remember DNA is POLAR!
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Nucleotides are joined through a phosphodiester
linkage between 5 and 3 carbon atoms. The 5
phosphate end is added to the 3 OH on the sugar.
Therefore, DNA synthesis proceeds from 5 to
3. Always. ALWAYS!!!!!!!!
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DNA polymerase catalyses the addition of
nucleotides to the 3OH end of the growing DNA
strand. DNA polymerase works ONLY in 5 to 3
direction.
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DNA Polymerase needs a Clamp to keep it stable.
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Because DNA polymerase works in only one
direction, the mechanism of replication differs
for the different strands of DNA
Leading strand synthesis is continuous Lagging
strand synthesis is discontinuous
(backstitching)
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DNA Primase Provides a Recognition Site for DNA
Polymerase during Lagging Strand Synthesis
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DNA Helicase unwinds the double helix
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Single-strand DNA binding proteins stabilize DNA
after unwinding of the helix
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The (almost) complete picture of DNA synthesis
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What else can DNA do?

• DNA can repair itself avoid mutations
• DNA can recombine genetic diversity
• DNA can JUMP! Transposons

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DNA Repair 1 replacing a single base
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DNA Repair 2 Replacing a sequence of nucleotides
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Homologous Recombination
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• The process begins at the site of a
  double-strand break in the chromosome
• An exonuclease creates 3 overhangs
• These free 3 ends can find homologous regions
  in a second chromosome
• DNA synthesis proceeds
• Selective strand cuts produce two chromosomes
  that have crossed over.

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Transposons Mobile Genetic Elements (Jumping
Genes)
DNA sequences that can insert themselves into
many different sites. Barbara McClintock was
awarded the Nobel Prize in 1983 for the discovery
of transposons.
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Suggested reading
Dynamic in vivo imaging and cell tracking using a
histone fluorescent fusion protein in mice.
Hadjantonakis AK and Papaioannou VE 2004 BMC
Biotechnol 433 The dynamics of histone H1
function in chromatin. Bustin M,Catez,F, Lim JH
2005 Mol. Cell17(5)617-620 Visualizing
polynucleotide polymerase machines at work.
Steitz TA 2006 EMBO J. 25(15) 3458-3468 (pretty
pictures of polymerases!)
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Reading for the next lecture
Imaging cerebral gene transcripts in live
animals Liu CH, Kim YR, Ren JQ, Eichler F, Rosen
BR, Liu PK J. Neurosci. 27(3) 713-722, 2007

• Discussion points
• What is the model?
• What is the imaging modality? Why was this
  modality chosen?
• What is the probe? How was it designed?
• Could they image transcription?