Chapter 16~ The Molecular Basis of Inheritance

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Chapter 16 The Molecular Basis of Inheritance
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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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The Transforming Principle
1928

• Frederick Griffith
• Streptococcus pneumonia bacteria
• was working to find cure for pneumonia
• harmless live bacteria (rough) mixed with
  heat-killed pathogenic bacteria (smooth) causes
  fatal disease in mice
• a substance passed from dead bacteria to live
  bacteria to change their phenotype
• Transforming Principle

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The Transforming Principle
mix heat-killed pathogenic non-pathogenic bact
eria
live pathogenic strain of bacteria
live non-pathogenic strain of bacteria
heat-killed pathogenic bacteria
A.
B.
D.
C.
mice die
mice live
mice live
mice die
Transformation change in phenotype something in
heat-killed bacteria could still transmit
disease-causing properties
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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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Avery, McCarty MacLeod
1944 ??!!

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

Oswald Avery
Maclyn McCarty
Colin MacLeod
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Confirmation of DNA
1952 1969 Hershey

• Hershey Chase
• classic blender experiment
• worked with bacteriophage
• viruses that infect bacteria
• grew phage viruses in 2 media, radioactively
  labeled with either
• 35S in their proteins
• 32P in their DNA
• infected bacteria with labeled phages

Why useSulfurvs.Phosphorus?
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Hershey Chase
Protein coat labeled with 35S
DNA labeled with 32P
T2 bacteriophages are labeled with radioactive
isotopes S vs. P
bacteriophages infect bacterial cells
bacterial cells are agitated to remove viral
protein coats
Which radioactive marker is found inside the cell?
Which molecule carries viral genetic info?
32P radioactivity foundin the bacterial cells
35S radioactivity found in the medium
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Blender experiment

• Radioactive phage bacteria in blender
• 35S phage
• radioactive proteins stayed in supernatant
• therefore viral protein did NOT enter bacteria
• 32P phage
• radioactive DNA stayed in pellet
• therefore viral DNA did enter bacteria
• Confirmed DNA is transforming factor

Taaa-Daaa!
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Hershey Chase
1952 1969 Hershey
Alfred Hershey
Martha Chase
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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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Structure of DNA
1953 1962

• Watson Crick
• developed double helix model of DNA
• other leading scientists working on question
• Rosalind Franklin
• Maurice Wilkins
• Linus Pauling

Wilkins
Pauling
Franklin
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1953 article in Nature
Watson and Crick
Crick
Watson
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Rosalind Franklin (1920-1958)
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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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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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The DNA backbone
5?
PO4

• Putting the DNA backbone together
• refer to the 3? and 5? ends of the DNA
• the last trailing carbon

base
CH2
5?
O
4?
1?
C
3?
2?
O
P
O
O
Sounds trivial, butthis will beIMPORTANT!!
O
base
CH2
5?
O
1?
4?
2?
3?
OH
3?
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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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Bonding in DNA
5?
3?
3?
5?
.strong or weak bonds? How do the bonds fit the
mechanism for copying DNA?
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Base pairing in DNA

• Purines
• adenine (A)
• guanine (G)
• Pyrimidines
• thymine (T)
• cytosine (C)
• Pairing
• A T
• 2 bonds
• C G
• 3 bonds

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But how is DNA copied?

• Replication of DNA
• base pairing suggests that it will allow each
  side to serve as a template for a new strand

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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Copying DNA

• Replication of DNA
• base pairing allows each strand to serve as a
  template for a new strand
• new strand is 1/2 parent template 1/2 new DNA
• semi-conservative copy process

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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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DNA Replication
Lets meetthe team

• Large team of enzymes coordinates replication

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Replication 1st step

• Unwind DNA
• helicase enzyme
• unwinds part of DNA helix
• stabilized by single-stranded binding proteins

helicase
single-stranded binding proteins
replication fork
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Replication 2nd step

• Build daughter DNA strand
• add new complementary bases
• DNA polymerase III

DNA Polymerase III
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Replication
3?
5?
DNA Polymerase III
energy

• Adding bases
• can only add nucleotides to 3? end of a growing
  DNA strand
• need a starter nucleotide to bond to
• strand only grows 5??3?

DNA Polymerase III
energy
DNA Polymerase III
energy
DNA Polymerase III
energy
3?
5?
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Leading Lagging strands

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

?
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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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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DNA polymerases

• DNA polymerase III
• 1000 bases/second!
• main DNA builder
• DNA polymerase I
• 20 bases/second
• editing, repair primer removal

DNA polymerase III enzyme
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Editing proofreading DNA

• 1000 bases/second lots of typos!
• DNA polymerase I
• proofreads corrects typos
• repairs mismatched bases
• removes abnormal bases
• repairs damage throughout life
• reduces error rate from 1 in 10,000 to 1 in 100
  million bases

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Fast accurate!

• It takes E. coli lt1 hour to copy 5 million base
  pairs in its single chromosome
• divide to form 2 identical daughter cells
• Human cell copies its 6 billion bases divide
  into daughter cells in only few hours
• remarkably accurate
• only 1 error per 100 million bases
• 30 errors per cell cycle

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What does it really look like?
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Any Questions??