DNA (Deoxyribonucleic Acid)

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DNA (Deoxyribonucleic Acid)
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Scientific History

• The march to understanding that DNA is the
  genetic material
• T.H. Morgan (1908)
• Frederick Griffith (1928)
• Avery, McCarty MacLeod (1944)
• Erwin Chargaff (1947)
• Hershey Chase (1952)
• Watson Crick (1953)
• Meselson Stahl (1958)

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Transformation of Bacteria
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What carries hereditary information?

• By the 1940s, scientists knew that chromosomes
  carried genes.
• They also knew that chromosomes were made of DNA
  and protein.
• They did NOT know which of these molecules
  actually carried the genes.
• Since protein has 20 types of amino acids that
  make it up, and DNA only has 4 types of building
  blocks, it was a logical conclusion.
• Most Scientists thought protein carried genes

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Chromosomes are made of DNA and protein
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Transformation of Bacteria
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DNA is the Transforming Principle
1944

• Avery, McCarty MacLeod
• purified both DNA proteins separately from
  Streptococcus pneumonia bacteria
• which will transform non-pathogenic bacteria?
• injected protein into bacteria
• no effect
• injected DNA into bacteria
• transformed harmless bacteria into virulent
  bacteria

mice die
Whats the conclusion?
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Averys Experiment
1. Avery repeated Griffiths experiments with an
additional step to see what type of molecule
caused transformation.
3. When Avery added enzymes that destroy DNA, no
transformation occurred.
Sohe knew that DNA carried hereditary
information!
2. Avery used enzymes to destroy the sugars and
transformation still occurredSugar did not cause
transformation. Avery used enzymes to destroy
lipids, RNA, and protein one by one. Every time
transformation still occurrednone of these had
anything to do with the transformation.
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Avery, McCarty MacLeod
1944 ??!!

• Conclusion
• first experimental evidence that DNA was the
  genetic material

Oswald Avery
Maclyn McCarty
Colin MacLeod
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Hershey-Chase Experiment

• The experiment involved viruses to see if DNA or
  protein was injected into the bacteria in order
  to make new viruses.
• One group of viruses was infected with
  radioactive protein and another group with
  radioactive DNA.
• Then the viruses attack the bacteria.
• Radioactive DNA shows up in the bacteria, but no
  radioactive protein.

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Chargaff
1947

• DNA composition Chargaffs rules
• varies from species to species
• all 4 bases not in equal quantity
• bases present in characteristic ratio
• humans
• A 30.9
• T 29.4
• G 19.9
• C 19.8

RulesA T C G
Thats interesting!What do you notice?
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Rosalind Franklin

• Took X-ray pictures of DNA.
• The photos revealed the basic helix, spiral shape
  of DNA.

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Maurice Wilkins

• Worked with Rosalind Franklin.
• Took her x-ray photos and information to Watson
  and Crick

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Watson and Crick

• Used Franklins pictures to build a series of
  large models.

• Stated that DNA is a double-stranded molecule in
  the shape of a double helix, or twisted ladder.
• Won the Nobel Prize for their work in 1962.

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Semiconservative replication,

• when a double helix replicates each of the
  daughter molecules will have one old strand and
  one newly made strand.
• Experiments in the late 1950s by Matthew Meselson
  and Franklin Stahl supported the semiconservative
  model, proposed by Watson and Crick, over the
  other two models. (Conservative dispersive)

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Double helix structure of DNA
It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material. Watson Crick
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Basic DNA Structure
P

• A nucleotide is the monomer of DNA
• A nucleotide is made of
• a sugar called deoxyribose
• a phosphate
• and a base (ATCG)

S
A
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Directionality of DNA

• You need to number the carbons!
• it matters!

nucleotide
PO4
N base
CH2
5?
This will beIMPORTANT!!
O
1?
4?
ribose
3?
2?
OH
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Deoxyribose

• Simple sugar molecule like glucose that has 5
  carbons
• The five carbons are numbered clockwise starting
  from the first one after the oxygen

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Phosphate

• The negatively charged phosphate bonds to the 5
  Carbon of the deoxyribose.

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Bases

• The base bonds to the 1 Carbon.

Base
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Bases

• There are two main types of bases purines and
  pyrimidines.
• Purines have two rings in their structure.
• Adenine and guanine are purines.
• Pyrimidines only have one ring.
• Thymine and Cytosine are pyrimidines.

Purines
Pyrimidines
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Basic DNA Structure
P

• To form one strand of DNA, the phosphate of one
  nucleotide covalently bonds to the 3 Carbon of
  the deoxyribose from another nucleotide.

S
A
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P
S
A

• The two strands of DNA are held together by
  hydrogen bonds

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Anti-parallel strands

• Nucleotides in DNA backbone are bonded from
  phosphate to sugar between 3? 5? carbons
• DNA molecule has direction
• complementary strand runs in opposite direction

5?
3?
3?
5?
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Base Pairs

• The nucleotides that bond together by their bases
  are called base pairs.
• Adenine only bonds to Thymine
• Guanine only bonds to Cytosine

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Does each of your cells have the same DNA?

• YES

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DNA Replication

• Before a cell divides, DNA must make a copy of
  itself so that each new cell has a complete set
  of DNA.

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Step 1-Unzip DNA

• An enzyme called helicase untwists the ladder and
  breaks the hydrogen bonds between the bases and
  unzips DNA down the middle.

Helicase Enzyme
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Step 2-Prime the DNA

• An enzyme called DNA primase put a few
  nucleotides of RNA on the DNA.
• This is only to create a starting place and these
  will later be removed.

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Step 3-Elongation

• The two strands of the Parent DNA become
  templates for the new strands.
• New nucleotides are added by an enzyme called DNA
  polymerase.

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Step 3-Elongation

• DNA polymerase only adds nucleotides in the 5 to
  3 direction on both strands beginning at the RNA
  primer.

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Step 4 Fine tuning

• RNA primer is removed and any gaps are sealed by
  an enzyme called ligase.
• DNA polymerase proof reads the new copy and fixes
  any mistakes.

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Helicase unwinds and unzips DNA
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DNA Polymerase Adds New Nucleotides
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• Are the two copies of DNA the same?
• Why would it be important for the two copies of
  DNA to be the same?

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Leading Lagging strands

• Limits of DNA polymerase III
• can only build onto 3? end of an existing DNA
  strand

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Okazaki fragments
Lagging strand
growing replication fork
Leading strand
?

• Lagging strand
• Okazaki fragments
• joined by ligase
• spot welder enzyme

DNA polymerase III

• Leading strand
• continuous synthesis

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Replication fork / Replication bubble
leading strand
lagging strand
leading strand
lagging strand
leading strand
lagging strand
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Starting DNA synthesis RNA primers

• Limits of DNA polymerase III
• can only build onto 3? end of an existing DNA
  strand

growing replication fork
primase
RNA

• RNA primer
• built by primase
• serves as starter sequence for DNA polymerase III

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Starting DNA synthesis RNA primers

• Limits of DNA polymerase III
• can only build onto 3? end of an existing DNA
  strand

growing replication fork
primase
RNA

• RNA primer
• built by primase
• serves as starter sequence for DNA polymerase III

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Replacing RNA primers with DNA

• DNA polymerase I
• removes sections of RNA primer and replaces with
  DNA nucleotides

DNA polymerase I
growing replication fork
RNA
But DNA polymerase I still can only build onto 3?
end of an existing DNA strand
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Chromosome erosion
Houston, we have a problem!
All DNA polymerases can only add to 3? end of an
existing DNA strand
DNA polymerase I
growing replication fork
DNA polymerase III
RNA

• Loss of bases at 5? ends in every replication
• chromosomes get shorter with each replication
• limit to number of cell divisions?

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Telomeres

• Repeating, non-coding sequences at the end of
  chromosomes protective cap
• limit to 50 cell divisions

growing replication fork
telomerase

• Telomerase
• enzyme extends telomeres
• can add DNA bases at 5? end
• different level of activity in different cells
• high in stem cells cancers -- Why?

TTAAGGG
TTAAGGG
TTAAGGG
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Replication fork
DNA polymerase III
lagging strand
DNA polymerase I
3
primase
Okazaki fragments
5
5
ligase
SSB
3
5
3
helicase
DNA polymerase III
5
leading strand
3
SSB single-stranded binding proteins
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Length of DNA

• The length of the DNA from one cell is
• 3 meters
• "Unravel your DNA and it would stretch from here
  to the moon"

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DNA Packing
DNAdoublehelix(2-nmdiameter
Histones
Beads ona string
Nucleosome(10-nm diameter)
Tight helical fiber(30-nm diameter)
Supercoil(200-nm diameter)
700nm
Metaphase chromosome
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Nucleosomes
8 histone molecules

• Beads on a string
• 1st level of DNA packing
• histone proteins
• 8 protein molecules
• positively charged amino acids
• bind tightly to negatively charged DNA

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DNA packing as gene control

• Degree of packing of DNA regulates transcription
• tightly wrapped around histones
• no transcription
• genes turned off

• heterochromatin
• darker DNA (H) tightly packed
• euchromatin
• lighter DNA (E) loosely packed

H
E
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The End!