chapter 16~ the molecular basis of inheritance
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Chapter 16~ The Molecular Basis of Inheritance. 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) - PowerPoint PPT PresentationTRANSCRIPT
Chapter 16~ The Molecular Basis of Inheritance
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)
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”
The “Transforming Principle”
Transformation = change in phenotypesomething in heat-killed bacteria could still transmit disease-causing properties
live pathogenicstrain of bacteria
live non-pathogenicstrain of bacteria
mice die mice live
heat-killed pathogenic bacteria
mix heat-killed pathogenic & non-pathogenicbacteria
mice live mice die
A. B. C. D.
DNA is the “Transforming Principle”
• 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
1944
What’s theconclusion?
mice die
Oswald Avery Maclyn McCarty Colin MacLeod
Avery, McCarty & MacLeod• Conclusion
– first experimental evidence that DNA was the genetic material
1944 | ??!!
Confirmation of DNA• 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
1952 | 1969Hershey
Why useSulfurvs.Phosphorus?
Protein coat labeledwith 35S DNA labeled with 32P
bacteriophages infectbacterial cells
T2 bacteriophagesare labeled withradioactive isotopesS vs. P
bacterial cells are agitatedto remove viral protein coats
35S radioactivityfound in the medium
32P radioactivity foundin the bacterial cells
Which radioactive marker is found inside the cell?
Which molecule carries viral genetic info?
Hershey &
Chase
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!
Hershey & Chase
Alfred HersheyMartha Chase
1952 | 1969Hershey
Chargaff
• DNA composition: “Chargaff’s 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%
1947
That’s interesting!What do you notice?
RulesA = TC = G
Structure of DNA• Watson & Crick
– developed double helix model of DNA• other leading scientists working on question:
– Rosalind Franklin– Maurice Wilkins– Linus Pauling
1953 | 1962
Franklin Wilkins Pauling
Watson and Crick 1953 article in Nature
CrickWatson
Rosalind Franklin (1920-1958)
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
Directionality of DNA• You need to
number the carbons!– it matters!
OH
CH2O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
The DNA backbone• Putting the DNA
backbone together– refer to the 3 and 5
ends of the DNA• the last trailing carbon
OH
O
3
PO4
base
CH2O
base
OPO
C
O–O
CH2
1
2
4
5
1
2
3
3
4
5
5
Sounds trivial, but…this will beIMPORTANT!!
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
3
5
5
3
Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalentphosphodiesterbonds
hydrogenbonds
Base pairing in DNA• Purines
– adenine (A)– guanine (G)
• Pyrimidines– thymine (T)– cytosine (C)
• Pairing– A : T
• 2 bonds– C : G
• 3 bonds
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
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
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)
DNA Replication • Large team of enzymes coordinates replication
Let’s meetthe team…
Replication: 1st step• Unwind DNA
– helicase enzyme• unwinds part of DNA helix• stabilized by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
DNAPolymerase III
Replication: 2nd step Build daughter DNA
strand add new
complementary bases DNA polymerase III
• Adding bases – can only add
nucleotides to 3 end of a growing DNA strand• need a “starter”
nucleotide to bond to
– strand only grows 53
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
energy
energy
energy
Replicationenergy
3
3
5
5
Limits of DNA polymerase III can only build onto 3 end of an
existing DNA strand
Leading & Lagging strands
5
5
5
5
3
3
3
53
53 3
Leading strand
Lagging strandOkazaki fragments
ligase
Okazaki
Leading strand continuous synthesis
Lagging strand Okazaki fragments joined by ligase
“spot welder” enzyme
DNA polymerase III
3
5
growing replication fork
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
53
3
5
5lagging strand
5 3
DNA polymerase III
RNA primer built by primase serves as starter sequence for DNA
polymerase III
Limits of DNA polymerase III can only build onto 3 end of an
existing DNA strand
Starting DNA synthesis: RNA primers
5
5
5
3
3
3
5
3 53 5 3
growing replication fork primase
RNA
DNA polymerase I removes sections of RNA primer and
replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3 end of an existing DNA strand
Replacing RNA primers with DNA
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
ligase
Loss of bases at 5 ends in every replication
chromosomes get shorter with each replication limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can only add to 3 end of an existing DNA strand
Chromosome erosion
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
Houston, we have a problem!
Repeating, non-coding sequences at the end of chromosomes = protective cap
limit to ~50 cell divisions
Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells
high in stem cells & cancers -- Why?
telomerase
Telomeres
5
5
5
5
3
3
3
3
growing replication fork
TTAAGGGTTAAGGGTTAAGGG
Replication fork
3’
5’3’
5’
5’
3’3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA polymerase III
DNA polymerase III
DNA polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB
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
Arthur Kornberg1959
Roger Kornberg2006
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
Fast & accurate!• It takes E. coli <1 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
1
2
3
4
What does it really look like?
2007-2008
Any Questions??