DNA (Deoxyribonucleic Acid) Scientific History n The march to understanding that DNA is the genetic...
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Transcript of DNA (Deoxyribonucleic Acid) Scientific History n The march to understanding that DNA is the genetic...
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)
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
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
Avery’s Experiment1. Avery repeated Griffith’s experiments with an additional step to see what type of molecule caused transformation.
3. When Avery added enzymes that destroy
DNA, no transformation occurred.
2. Avery used enzymes to destroy the sugars and transformation still occurred—Sugar did not cause transformation. Avery used enzymes to destroy lipids, RNA, and protein one by one. Every time transformation still occurred—none of these had anything to do with the transformation.
So…he knew that DNA carried hereditary
information!
Oswald Avery Maclyn McCarty Colin MacLeod
Avery, McCarty & MacLeod Conclusion
– first experimental evidence that DNA was the genetic material
1944 | ??!!
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.
Hershey-Chase Experiment
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
Rosalind Franklin Took X-ray
pictures of DNA. The photos
revealed the basic helix, spiral shape of DNA.
Maurice Wilkins Worked with
Rosalind Franklin. Took her x-ray
photos and information to Watson and Crick
Watson and Crick
Used Franklin’s 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.
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)
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
Basic DNA Structure
S
P
A
CS
P
S
P
T
A nucleotide is the monomer of DNA A nucleotide is made of
– a sugar called deoxyribose– a phosphate– and a base (ATCG)
Directionality of DNA You need to
number the carbons!– it matters!
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
Deoxyribose
Simple sugar molecule like glucose that has 5 carbons
The five carbons are numbered clockwise starting from the first one after the oxygen
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.
Pyrimidines Purines
Basic DNA Structure
S
P
A
CS
P
S
P
T
To form one strand of DNA, the phosphate of one nucleotide covalently bonds to the 3’ Carbon of the deoxyribose from another nucleotide.
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
Base Pairs The nucleotides that bond together by
their bases are called base pairs.– Adenine only bonds to Thymine– Guanine only bonds to Cytosine
DNA Replication
Before a cell divides, DNA must make a copy of itself so that each new cell has a complete set of DNA.
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
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.
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.
Step 3-Elongation
DNA polymerase only adds nucleotides in the 5’ to 3’ direction on both strands beginning at the RNA primer.
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.
DNA Polymerase Adds New Nucleotides
T
G S
S
P
P
S
P
A
S
P
A
CS
P
S
P
T
T S
P
G S
P
S
P
A
S
P
A
CS
P
S
P
T
Are the two copies of DNA the same?
Why would it be important for the two copies of DNA to be the same?
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 strand
Okazaki 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 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
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"
DNA PackingDNAdoublehelix(2-nmdiameter
Histones“Beads ona string”
Nucleosome(10-nm diameter)
Tight helical fiber(30-nm diameter) Supercoil
(200-nm diameter)
Metaphase chromosome
700nm
Nucleosomes “Beads on a string”
– 1st level of DNA packing– histone proteins
• 8 protein molecules• positively charged amino acids • bind tightly to negatively charged DNA
8 histone molecules
DNA packing as gene control Degree of packing of DNA regulates transcription
– tightly wrapped around histones • no transcription
• genes turned off heterochromatindarker DNA (H) = tightly packed
euchromatinlighter DNA (E) = loosely packed
H E