Transcript Lecture #7 Date - clevengerscience
DNA (Deoxyribonucleic Acid)
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)
Transformation of Bacteria
What carries hereditary information?
chromosomes carried genes.
They also knew that chromosomes were made of DNA and protein.
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
Most Scientists thought protein carried genes
Chromosomes
are made of
DNA
and
protein
Transformation of Bacteria
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 • no effect into bacteria – injected DNA into bacteria • transformed harmless bacteria into virulent bacteria
1944 mice die What ’ s the conclusion?
Avery’s Experiment
1. 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.
So…he knew that DNA carried hereditary information!
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.
1944 | ??!!
Avery, McCarty & MacLeod
Conclusion – first experimental evidence that DNA was the genetic material
Oswald Avery Maclyn McCarty Colin MacLeod
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.
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%
Rules A = T C = G
T = 29.4% G = 19.9%
That ’
C = 19.8%
What do you notice?
1947
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
P P P S S S Basic DNA Structure A C 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!
PO 4 5
CH 2 This will be IMPORTANT!! 4
O nucleotide ribose N base 1
3
OH 2
Deoxyribose
Simple sugar molecule like glucose that has 5 carbons The five carbons are numbered clockwise starting from the first one after the oxygen
Phosphate
The negatively charged phosphate bonds to the 5’ Carbon of the deoxyribose.
Bases
The base bonds to the 1’ Carbon.
Base
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
P P P S S S Basic DNA Structure A C T
To form one strand of DNA, the phosphate of one nucleotide covalently bonds to the 3’ Carbon of the deoxyribose from another nucleotide.
P S A T S P S C G S P S T A S
The two strands of DNA are held together by
hydrogen bonds P P P
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
Base Pairs
The nucleotides that bond together by their bases are called base pairs.
– Adenine only bonds to Thymine – Guanine only bonds to Cytosine
Does each of your cells have the same DNA?
YES
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.
Helicase unwinds and unzips DNA P S A T S P P S C G S P P S T A S P
DNA Polymerase Adds New Nucleotides P S A P S C P S T T S P G S A S P P P S A P S C P S T T S P G S A S P P
Are the two copies of DNA the same?
Why would it be important for the two copies of DNA to be the same?
Okazaki
Leading & Lagging strands
Limits of DNA polymerase III
can only build onto 3
end of an existing DNA strand 5
5
5
3
Lagging strand 3
5
3
5
3
ligase growing replication fork 3
5
Leading strand 3
5
3
Lagging strand
Okazaki fragments joined by ligase
“spot welder” enzyme DNA polymerase III Leading strand
continuous synthesis
3
5
3
5
5
3
Replication fork / Replication bubble
5
3
DNA polymerase III 3
leading strand 5
5
3
5
lagging strand 5
3
5
3 lagging strand
5
growing replication fork 3
leading strand 5
leading strand lagging strand 5
5
5
3
5
growing replication fork 5
3
Starting DNA synthesis: RNA primers
Limits of DNA polymerase III
can only build onto 3
end of an existing DNA strand 3
5
3
5
3
5
growing replication fork 3
5
DNA polymerase III RNA primer
built by primase serves as starter sequence for DNA polymerase III 5
3
primase RNA 5
3
Starting DNA synthesis: RNA primers
Limits of DNA polymerase III
can only build onto 3
end of an existing DNA strand 3
5
3
5
3
5
growing replication fork 3
5
DNA polymerase III RNA primer
built by primase serves as starter sequence for DNA polymerase III 5
3
primase RNA 5
3
Replacing RNA primers with DNA
DNA polymerase I
removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I 5
3
3
5
growing replication fork 3
ligase 5
RNA 5
3
But DNA polymerase I still can only build onto 3
end of an existing DNA strand
Houston, we have a problem!
Chromosome erosion
All DNA polymerases can only add to 3
end of an DNA polymerase I existing DNA strand 3
5
5
growing replication fork 3
DNA polymerase III Loss of bases at 5
ends in every replication
chromosomes get shorter with each replication limit to number of cell divisions?
RNA 5
3
5
3
Telomeres
Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions
3
5
5
growing replication fork 3
Telomerase
enzyme extends telomeres can add DNA bases at 5
end different level of activity in different cells
high in stem cells & cancers -- Why?
5
3
telomerase 5
TTAAGGG TTAAGGG 3
Replication fork
DNA polymerase III lagging strand DNA polymerase I primase Okazaki fragments 5’ 3’ ligase 5’ SSB 3’ 5’ 3’ 3’ 5’ helicase DNA polymerase III leading strand direction of replication SSB = single-stranded binding proteins
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 Packing
Histones “Beads on a string” DNA double helix (2-nm diameter Nucleosome (10-nm diameter) Tight helical fiber (30-nm diameter ) 700 nm Metaphase chromosome Supercoil (200-nm diameter )
Nucleosomes
“Beads on a string” – 1 st 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
heterochromatin darker DNA (H) = tightly packed euchromatin lighter DNA (E) = loosely packed H E