Download The Search for the Genetic Material

Chapter 16
The Molecular Basis
of Inheritance
Question?
• Traits are inherited on
chromosomes, but what in the
chromosomes is the genetic
material?
• Two possibilities:
• Protein
• DNA
Qualifications
• Protein:
• very complex.
• high specificity of function.
• DNA:
• simple.
• not much known about it (early
1900’s).
For testing:
• Name(s) of experimenters
• Outline of the experiment
• Result of the experiment and
the importance of the result
Griffith - 1928
• Pneumonia in mice.
• Two strains:
• S - pathogenic
• R - harmless
Griffith’s Experiment
Result
• Something turned the R cells
into S cells.
• Transformation - the
assimilation of external genetic
material by a cell.
Problem
• Griffith used heat.
• Heat denatures proteins.
• So could proteins be the
genetic material?
• DNA - heat stable.
• Griffith’s results contrary to
accepted views.
Avery, McCarty and MacLeod
- 1944
• Repeated Griffith’s experiments,
but added specific fractions of S
cells.
• Result - only DNA transformed
R cells into S cells.
• Result - not believed.
Hershey & Chase -1952
• Genetic information of a virus or
phage.
• Phage - virus that attacks
bacteria and reprograms host to
produce more viruses.
Bacteria with Phages
Phage Components
• Two main chemicals:
• Protein
• DNA
• Which material is transferred to
the host?
Used Tracers
• Protein - CHONS, can trace
with 35S.
• DNA - CHONP, can trace with
32P.
Experiment
• Used phages labeled with one
tracer or the other and looked to
see which tracer entered the
bacteria cells.
Result
• DNA enters the host cell, but
the protein did not.
• Therefore:
DNA is the genetic material.
Picture Proof
Chargaff - 1947
• Studied the chemical
composition of DNA.
• Found that the nucleotides were
in certain ratios.
Chargaff’s Rule
• A=T
• G=C
• Example: in humans,
A = 30.9%
T = 29.4%
G = 19.9%
C = 19.8%
Why?
• Not known until Watson and
Crick worked out the structure
of DNA.
Watson and Crick - 1953
• Used X-ray crystallography data
(from Rosalind Franklin)
• Used model building.
• Result - Double Helix Model of
DNA structure.
• (One page paper, 1953).
Rosalind Franklin
Book & Movies
• “The Double Helix” by James
Watson- His account of the
discovery of the shape of DNA
• Movie – The Double Helix
DNA Composition
• Deoxyribose Sugar (5-C)
• Phosphate
• Nitrogen Bases:
• Purines
• Pyrimidines
DNA Backbone
• Polymer of sugar-phosphate.
• 2 backbones present.
Nitrogen Bases
• Bridge the backbones together.
• Purine + Pyrimidine = 3 rings.
• Constant distance between the
2 backbones.
• Held together by H-bonds.
Chargaff’s Rule
• Explained by double helix
model.
• A = T, 3 ring distance.
• G = C, 3 ring distance.
Watson and Crick
• Published a second paper
(1954) that speculated on the
way DNA replicates.
• Proof of replication given by
others.
Replication
• The process of making more
DNA from DNA.
• Problem: when cells replicate,
the genome must be copied
exactly.
• How is this done?
Models for DNA Replication
• Conservative - one old strand,
one new strand.
• Semiconservative - each strand
is 1/2 old, 1/2 new.
• Dispersive - strands are
mixtures of old and new.
Replication Models
Meselson – Stahl, late 1950’s
• Grew bacteria on two isotopes
of N.
• Started on 15N, switched to
14N.
• Looked at weight of DNA after
one, then 2 rounds of
replication.
Results
• Confirmed the
Semiconservative Model of DNA
replication.
Replication - Preview
• DNA splits by breaking the Hbonds between the backbones.
• Then DNA builds the missing
backbone using the bases on
the old backbone as a template.
Origins of Replication
• Specific sites on the DNA
molecule that starts replication.
• Recognized by a specific DNA
base sequence.
Prokaryotic
• Circular DNA.
• 1 origin site.
• Replication runs in both
directions from the origin site.
Eukaryotic Cells
• Many origin sites.
• Replication bubbles fuse to form
new DNA strands.
DNA Elongation
• By DNA Polymerases such as
DNA pol III
• Adds DNA triphosphate
monomers to the growing
replication strand.
• Matches A to T and G to C.
Energy for Replication
• From the triphosphate
monomers.
• Loses two phosphates as each
monomer is added.
Problem of Antiparallel DNA
• The two DNA strands run
antiparallel to each other.
• DNA can only elongate in the
5’--> 3’ direction.
Leading Strand
• Continuous replication toward
the replication fork in the 5’-->3’
direction.
Leading Strand
• 1. DNA helicase unwinds the
DNA at the replication forks.
• -leading strand (3’-5’), replicates (5’-3’)
towards the fork.
2. Molecules of single strand binding
protein prevent the DNA from sticking
back together.
3. Primase synthesizes an RNA primer at
the end of 5’end.
4. DNA pol III synthesizes the strand
continuously.
Lagging Strand
• 1. DNA helicase unwinds the
DNA at the replication fork.
• -lagging strand (5’-3’) cannot replicate
in the 3’-5’ direction, replicates away
and towards the fork.
2. Primase joins RNA nucleotides into a
primer.
3. DNA pol III adds DNA nucleotides to
the primer forming an Okasaki fragment
1.
4. After reaching the next RNA primer
DNA pol III detaches.
• 5. Fragment 2 is primed, then
DNA pol III adds DNA
nucleotides, detaching when it
reaches the fragment 1 primer.
• 6. DNA pol I replaces the RNA
with DNA, adding nucleotides to
the 3’ end of fragment 2.
• 7. DNA ligase forms a bond
between the newest DNA and
the DNA of fragment 1.
• 8. This continues until the
strand is replicated.
Priming
• DNA pol III cannot initiate DNA
synthesis.
• Nucleotides can be added only
to an existing chain called a
Primer.
Primer
• Make of RNA.
• 10 nucleotides long.
• Added to DNA by an enzyme
called Primase.
• DNA is then added to the RNA
primer.
Priming
• A primer is needed for each
DNA elongation site.
Lagging Strand
• Discontinuous synthesis away
from the replication fork.
• Replicated in short segments as
more template becomes
opened up.
Okazaki Fragments
• Short segments (100-200
bases) that are made on the
lagging strand.
• All Okazaki fragments must be
primed.
• RNA primer is removed after
DNA is added.
Enzymes
• DNA pol I - replaces RNA
primers with DNA nucleotides.
• DNA Ligase - joins all DNA
fragments together.
Other Proteins in Replication
• Topoisomerase – relieves
strain ahead of replication
forks.
• Helicase - unwinds the DNA
double helix.
• Single-Strand Binding Proteins
- help hold the DNA strands
apart.
DNA Replication Error Rate
• 1 in 1 billion base pairs.
• About 3 mistakes in our DNA
each time it’s replicated.
Reasons for Accuracy
• DNA pol III self-checks and
corrects mismatches.
• DNA Repair Enzymes - a family
of enzymes that checks and
corrects DNA.
DNA Repair
• Over 130 different DNA repair
enzymes known.
• Failure to repair may lead to
Cancer or other health
problems.
Example:
• Xeroderma Pigmentosum Genetic condition where a DNA
repair enzyme doesn’t work.
• UV light causes damage, which
can lead to cancer.
Xeroderma Pigmentosum
Cancer
Protected from UV
Thymine Dimers
• T-T binding from side to side
causing a bubble in DNA
backbone.
• Often caused by UV light.
Excision Repair
• Cuts out the damaged DNA.
• DNA Polymerase fills in the
excised area with new bases.
• DNA Ligase seals the
backbone.
Problem - ends of DNA
• DNA Polymerase can only add
nucleotides in the 5’--->3’
direction.
• It can’t complete the ends of the
DNA strand.
Result
• DNA gets shorter and shorter
with each round of replication.
Telomeres
• Repeating units of TTAGGG
(100- 1000 X) at the end of the
DNA strand (chromosome)
• Protects DNA from unwinding
and sticking together.
• Telomeres shorten with each
DNA replication.
Telomeres
Telomeres
• Serve as a “clock” to count how
many times DNA has replicated.
• When the telomeres are too short,
the cell dies by apoptosis.
Implication
• Telomeres are involved with the
aging process.
• Limits how many times a cell line
can divide.
Telomerase
• Enzyme that uses RNA to
rebuild telomeres.
• Can make cells “immortal”.
• Found in cancer cells.
• Found in germ cells.
• Limited activity in active cells
such as skin cells
Comment
• Control of Telomerase may stop
cancer, or extend the life span.
NEWS FLASH
• The DNA of Telomers is
actually used to build proteins.
• These proteins seem to impede
telomerase.
• Feedback Loop??