Download Inheritance - Molecular Basis

DNA - The Molecular Basis of Inheritance
Important Early Discoveries
Fred Griffith (1928) – Experiments with pneumonia and
bacterial transformation determined that there is a molecule
that controls inheritance.
Oswald T. Avery (1944) - Transformation experiment
determined that DNA was the genetic material responsible
for Griffith’s results (not RNA).
Hershey-Chase Experiments (1952) – discovered that
DNA from viruses can program bacteria to make new
viruses.
Erwin Chargaff (1947) – noted that the the amount of
A=T and G=C and an overall regularity in the amounts of
A,T,C and G within species.
Frederick Griffith’s Transformation Experiment
•
•
•
•
The discovery of the genetic role of DNA began with research by Frederick
Griffith in 1928
Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a
harmless “R” strain
When he mixed heat-killed remains of the pathogenic strain with living cells of
the harmless strain, some living cells became pathogenic
He called this phenomenon transformation, now defined as a change in
genotype and phenotype due to assimilation of foreign DNA
Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells (control)
Mixture of heat-killed
S cells and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample
Oswald T. Avery’s Transformation Experiment
•
•
•
In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced
that the transforming substance was DNA
Their conclusion was based on experimental evidence that only DNA
worked in transforming harmless bacteria into pathogenic bacteria
Many biologists remained skeptical, mainly because little was known
about DNA
Life Cycle Of Virulent T2 Phage
Hershey-Chase Bacteriophage Experiment
•
•
•
•
In 1952, Alfred Hershey and Martha Chase performed experiments showing
that DNA is the genetic material of a phage known as T2
To determine the source of genetic material in the phage, they designed an
experiment showing that only one of the two components of T2 (DNA or
protein) enters an E. coli cell during infection
32P is discovered within the bacteria and progeny phages, whereas 35S is
not found within the bacteria but released with phage ghosts.
They concluded that the injected DNA of the phage provides the genetic
information
Phage
Radioactive
protein
Empty
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1:
Sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
Phosphorus (32P)
Centrifuge
Pellet
Radioactivity
(phage DNA)
in pellet
Additional Evidence That DNA Is the Genetic Material
• In 1947, Erwin Chargaff reported that DNA composition varies
from one species to the next
• This evidence of diversity made DNA a more credible candidate
for the genetic material
• By the 1950s, it was already known that DNA is a polymer of
nucleotides, each consisting of a nitrogenous base, a sugar, and
a phosphate group
• Franklin’s X-ray crystallographic images of DNA enabled
Watson to deduce that DNA was helical
• The X-ray images also enabled Watson to deduce the width of
the helix and the spacing of the nitrogenous bases
• The width suggested that the DNA molecule was made up of
two strands, forming a double helix
James D. Watson & Francis H. Crick
• In 1953 presented the double helix model of DNA
• Two primary sources of information:
– 1. Chargaff Rule: #A#T and #G#C. “A strange but
possibly meaningless phenomenon”.
– 2. X-ray diffraction studies of Rosalind Franklin & Maurice H.
F. Wilkins
DNA Structure
• Conclusion-DNA is a helical structure with distinctive
regularities, 0.34 nm & 3.4 nm.
1962: Nobel Prize in Physiology and Medicine
Watson, J.D. and F.H. Crick, “Molecular Structure of
Nucleic Acids: A Structure for Deoxynucleic Acids”. Nature
171 (1953), p. 738.
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
What about?
Rosalind Franklin
The Structure of DNA
• DNA is composed of four nucleotides,
each containing: adenine, cytosine,
thymine, or guanine.
• The amounts of A = T, G = C, and
purines = pyrimidines [Chargaff’s
Rule].
• DNA is a double-stranded helix with
antiparallel strands [Watson and Crick].
• Nucleotides in each strand are linked
by 5’-3’ phosphodiester bonds
• Bases on opposite strands are linked
by hydrogen bonding: A with T, and G
with C.
The Basic Principle: Base Pairing to a Template Strand
•
•
•
The relationship between structure and function is manifest in the
double helix
Since the two strands of DNA are complementary each strand acts as a
template for building a new strand in replication
In DNA replication, the parent molecule unwinds, and two new daughter
strands are built based on base-pairing rules
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
5 end
DNA replication
• The parent molecule unwinds, and two new
daughter strands are built based on basepairing rules
T
A
T
A
T
A
C
G
C
G
C
T
A
T
A
T
A
A
T
A
T
A
T
G
C
G
C
G
C
G
A
T
A
T
A
T
C
G
C
G
C
G
T
A
T
A
T
A
T
A
T
A
T
C
G
C
G
C
A
G
(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
DNA Replication
• DNA must replicate during each cell division
• 3 alternative models for DNA replication were hypothesized:
– Semiconservative replication
– Conservative replication
– Dispersive replication
Semi-conservative
Conservative
Dispersive
Meselson-Stahl Experiments
• Labeled the nucleotides of
old strands with a heavy
isotope of nitrogen (15N),
new nucleotides were
indicated by a lighter isotope
(14N).
• The first replication in the
14N medium produced a
band of hybrid (15N-14N)
DNA, eliminating the
conservative model.
• A second replication
produced both light and
hybrid DNA, eliminating the
dispersive model and
supporting the
semiconservative model.
Bacteria
cultured in
medium
containing
Bacteria
transferred to
medium
containing
15N
14N
DNA sample
centrifuged
after 20 min
(after first
replication)
DNA sample
centrifuged
after 40 min
(after second
replication)
First replication
Conservative
model
Semiconservative
model
Dispersive
model
Less
dense
More
dense
Second replication
DNA Replication is “Semi-conservative”
• Each 2-stranded
daughter molecule is
only half new
• One original strand was
used as a template to
make the new strand
DNA Replication
• The copying of DNA is remarkable in its speed and
accuracy
• Involves unwinding the double helix and synthesizing
two new strands.
• More than a dozen enzymes and other proteins
participate in DNA replication
• The replication of a DNA molecule begins at special
sites called origins of replication, where the two strands
are separated
Origins of Replication
• A eukaryotic chromosome may have hundreds or
even thousands of replication origins
Origin of replication
1 Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
Parental (template) strand
Daughter (new) strand
Bubble
0.25 µm
Replication fork
2 The bubbles expand laterally, as
DNA replication proceeds in both
directions.
3 Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete.
Two daughter DNA molecules
(a) In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome.
(b) In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
Mechanism of DNA Replication
• DNA replication is catalyzed by
DNA polymerase III which needs
an RNA primer
• DNA polymerase III cannot
initiate the synthesis of a
polynucleotide, they can only
add nucleotides to the 3 end
• The initial nucleotide strand is an
RNA primer
• RNA primase synthesizes primer
on DNA strand
• DNA polymerase adds
nucleotides to the 3’ end of the
growing strand
DNA polymerase III adds
nucleotides to primer
DNA polymerase I degrades
the RNA primer and
replaces it with DNA
Mechanism of DNA Replication
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•
•
•
•
Nucleotides are added by complementary base pairing with the template strand
DNA always reads from 5’ end to 3’ end for transcription replication
During replication, new nucleotides are added to the free 3’ hydroxyl on the
growing strand
The nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added,
releasing energy for DNA synthesis.
The rate of elongation is about 500 nucleotides per second in bacteria and 50
per second in human cells
New strand
5 end
Template strand
3 end
5 end
3 end
Sugar
Phosphate
Base
3 end
DNA polymerase
Pyrophosphate3 end
Nucleoside
triphosphate
5 end
5 end
The Mechanism of DNA Replication
• DNA synthesis on the leading
strand is continuous
• Only one primer is needed for
synthesis of the leading strand
• The lagging strand grows the
same general direction as the
leading strand (in the same
direction as the Replication
Fork). However, DNA is made
in the 5’-to-3’ direction
• Therefore, DNA synthesis on
the lagging strand is
discontinuous
• For synthesis of the lagging
strand, each fragment (Okazaki)
must be primed separately, then
DNA fragments are sythesized
and subsequently ligated
together
3
5
Parental DNA
Leading strand
5
3
Okazaki
fragments
Lagging strand
3
5
DNA pol III
Template
strand
Leading strand
Lagging strand
Template
strand
DNA ligase
Overall direction of replication
Mechanism of DNA Replication
•
Many proteins assist in DNA replication
– DNA helicases unwind the double helix, the template strands are stabilized
by other proteins
– Single-stranded DNA binding proteins make the template available
– RNA primase catalyzes the synthesis of short RNA primers, to which
nucleotides are added.
– DNA polymerase III extends the strand in the 5’-to-3’ direction
– DNA polymerase I degrades the RNA primer and replaces it with DNA
– DNA ligase joins the DNA fragments into a continuous daughter strand
Overall direction of replication
Leading
strand
DNA pol III
Replication fork
Primase
DNA pol III
Primer
5
3
Parental DNA
Lagging
strand
Leading
Origin of replication
strand
Lagging
strand
Lagging
strand
Leading
strand
OVERVIEW
DNA ligase
DNA pol I
3
5
Enzymes in DNA replication
Helicase unwinds
parental double helix
DNA polymerase III
binds nucleotides
to form new strands
Binding proteins
stabilize separate
strands
Primase adds
short primer
to template strand
DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
Replication
3’
3’
5’
5’
3’
5’
3’
5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
DNA polymerase III enzyme adds DNA nucleotides
to the RNA primer.
Replication
Overall direction
of replication
3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
3’
5’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’
5’
Replication
3’
5’
3’
5’
3’
5’
3’ 5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Exonuclease activity of DNA polymerase I
removes RNA primers.
Replication
3’
3’
5’
3’
5’
3’5’
3’
5’
Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.
Replication Fork Overview
Overall direction of replication
Leading
strand
Lagging
Leading
Origin of replication
strand
strand
Lagging
strand
DNA pol III
3
Parental DNA
DNA pol I
Primase
Primer
Lagging
strand
Leading
strand
DNA ligase
Replication fork
5
OVERVIEW
DNA pol III
3
5
Other Proteins That Assist DNA Replication
• Helicase, topoisomerase, single-strand binding
protein are all proteins that assist DNA replication
Proofreading
• DNA must be faithfully replicated…but
mistakes occur
– DNA polymerase (DNA pol) inserts the wrong
nucleotide base in 1/10,000 bases
• DNA pol has a proofreading capability and can correct
errors
– Mismatch repair: ‘wrong’ inserted base can be
removed
– Excision repair: DNA may be damaged by
chemicals, radiation, etc. Mechanism to cut out
and replace with correct bases
Mutations
• A mismatching of base pairs, can occur at a
rate of 1 per 100,000 bases.
• DNA polymerase proofreads and repairs
accidental mismatched pairs.
• Chances of a mutation occurring at any one
gene is over 1 in 10,000,000,000 (billion)
• Because the human genome is so large,
even at this rate, mutations add up. Each of
us probably inherited 3-4 mutations!
Proofreading and Repairing DNA
• DNA polymerases
proofread newly made
DNA, replacing any
incorrect nucleotides
• In mismatch repair of DNA,
repair enzymes correct
errors in base pairing
• In nucleotide excision DNA
repair nucleases cut out
and replace damaged
stretches of DNA
1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
DNA
polymerase
3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
4 DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
DNA repair
Accuracy of DNA Replication
• The chromosome of E. coli bacteria contains
about 5 million bases pairs
– Capable of copying this DNA in less than an hour
• The 46 chromosomes of a human cell contain
about 6 BILLION base pairs of DNA!!
– Printed one letter (A,C,T,G) at a time…would fill up
over 900 volumes of Campbell.
– Takes a cell a few hours to copy this DNA
– With amazing accuracy – an average of 1 per
billion nucleotides
Replicating the Ends of DNA Molecules
• The ends of eukaryotic chromosomal DNA get
shorter with each round of replication
5
Leading strand
Lagging strand
End of parental
DNA strands
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Shorter and shorter
daughter molecules
Telomeres
• Eukaryotic chromosomal DNA molecules
have at their ends nucleotide sequences,
called telomeres, that postpone the erosion of
genes near the ends of DNA molecules
1 µm
Telomerases
• If the chromosomes of germ cells became
shorter in every cell cycle essential genes
would eventually be missing from the gametes
they produce
• An enzyme called telomerase catalyzes the
lengthening of telomeres in germ cells