Download ch. 16 Molecular Basis of Inheritance-2009

Molecular Basis of Inheritance
Ch. 16
•DNA Structure
•DNA Replication
Evidence that DNA is the
Hereditary Material of Life?
• Griffith-Avery Experiment —DNA can
transform bacteria
• Hershey-Chase Experiment —Viral DNA
can program cells
• Chargaff —Analysis of DNA composition
Griffith Experiment
• Studied the bacterium
that caused pneumonia-S. pneumonia.
– (S) smooth cells produce
mucous capsules that
protect the bacteria from an
organism’s immune
system--pathogenic.
– (R) rough cells have no
mucous capsule and are
attacked by an organism’s
immune system--non
pathogenic.
http://www.bio.miami.edu/~cmallery/150/gene/sf11x1a.jpg
Griffith Experiment
http://www.nature.com/scitable/nated/content/18491/sadava_11_1_la
rge_2.gif
• Mixed heat-killed (S) bacteria with living (R) bacteria
• The new bacteria that arose from the bacteria were
somehow transformed into pathogenic S. pneumonia.
• Griffith called this process transformation.
Griffith’s Transformation Experiment
• Did not identify DNA as the transforming
factor, but it set the stage for other
experiments.
Oswaldt Avery
• Avery worked for a long time trying to
identify the transforming factor.
• After isolating and purifying numerous
macromolecules from the heat killed
pathogenic bacteria he and his colleagues
could only get DNA to work.
• The prevailing beliefs about proteins vs.
DNA continued to generate skepticism.
The Hershey-Chase Experiment
• In 1952, Alfred Hershey and Martha
Chase performed experiments with viruses
showing that DNA is genetic material.
• Viruses (aka phages) are DNA or RNA
wrapped in a protein.
• E. coli is a bacteria that is often used in
experiments.
http://course1.winona.edu/sberg/IMAGES/phage.GIF
The Hershey-Chase Experiment
• Used the T2 phage
because it was generally
accepted to be DNA
wrapped in protein.
• Used E. coli because it
was easily obtainable and
was readily attacked by
T2.
• Had to demonstrate
whether or it was DNA or
protein that was the
hereditary factor.
Hershey-Chase Experiment:
The Hershey-Chase Experiment
• They concluded:
– That the virus injects DNA into the E. coli and
it is the genetic material that programs the
cells to produce new T2 phages.
– The protein stays outside.
• This experiment provided firm evidence
that DNA was the hereditary material and
not protein.
Erwin Chargaff’s Experiment
• He discovered that the amount of adenine is
equal to the amount of thymine and cytosine
equaled the amount of guanine.
Adenine = 30.9%
Thymine = 29.4%
Guanine = 19.9%
Cytosine = 19.8%
• Chargaff did not know what all of this meant, but
after the elucidation of the shape of the DNA
molecule, these became known as Chargaff’s
Rules.
Linus Pauling
James Watson
Francis Crick
Maurice Wilkins
Rosalind Franklin
Linus Pauling
Rosalind Franklin
http://media.photobucket.com/image/wilkins%20and%20franklin/PhotozOnline/crickwatsonwilkins
http://37days.typepad.com/37days/images/2008/03/0
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•
•
•
•
•
http://www.achievement.org/achievers/pau0/large/p
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Scientists in the Race for the
Double Helix
• Maurice Wilkins and Rosalind Franklin used X-ray
crystallography to study the structure of DNA.
– X-rays are diffracted as they passed through
aligned fibers of purified DNA.
– The diffraction pattern is used to deduce the
three-dimensional shape of molecules.
Fig. 16.4
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Watson and Crick
• In 1953, James
Watson and Francis
Crick visited a lab of
Maurice Wilkins.
• Examined lab data
(an X-ray diffraction
image of DNA)
produced by Rosalind
Franklin.
http://eggbirdinc.com/images/watson_crick_500.jpg
Trial & Error using molecular models
made of wire:
• first tried to place the
sugar-phosphate chains
on the inside.
– Didn’t fit X-ray
measurements and
other information on
the chemistry of DNA.
• then put the sugarphosphate chain on the
outside and the nitrogen
bases on the inside of the
double helix.
http://www.gordon-ermer.com/uploaded_images/watson-702693.jpg
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The nitrogenous bases are paired in specific
combinations: adenine with thymine and
guanine with cytosine.
• Pairing like nucleotides did not fit the uniform
diameter indicated by the X-ray data.
– A purine-purine pair would be too wide and a
pyrimidine-pyrimidine pairing would be too short.
– Only a pyrimidinepurine pairing would
produce the 2-nm
diameter indicated
by the X-ray data.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Structure of DNA
•The phosphate group of one
nucleotide is attached to the
sugar of the next nucleotide
in line.
•The result is a “backbone” of
alternating phosphates and
sugars, from which the bases
project.
Purines
Adenine
Pyrimidines
Thymine
Guanine
Cytosine
• Watson and Crick determined that chemical
side groups off the nitrogen bases would
form hydrogen bonds, connecting the two
strands.
– Adenine forms two
hydrogen bonds
only with thymine
– Guanine forms three
hydrogen bonds only with
cytosine.
– This finding explained
Chargaff’s rules.
Fig. 16.6
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Purine + Pyrimidine:
width consistent with
X-ray data,
• base ratios
consistent with
Chargaff’s rules:
A = T and G  C
Structure of DNA is related to 2 primary functions:
1. Copy itself exactly for new cells that are created
2. Store and use information to direct cell activities
DNA Replication Models
Fig. 16.8
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Meselson and Stahl Experiment
• Supported the semiconservative model,
proposed by Watson and Crick, over the other
two models.
– In their experiments, they labeled the nucleotides of
the old strands with a heavy isotope of nitrogen (15N),
while any new nucleotides were indicated by a lighter
isotope (14N).
– Replicated strands could be separated by density in a
centrifuge.
– Each model-the semiconservative model, the
conservative model, and the dispersive model-made
specific predictions on the density of replicated DNA
strands.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• 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.
Fig. 16.9
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
DNA Replication
• Begins at a site
called the origin of
replication.
• Prokaryotes have
one origin of
replication.
• Eukaryotes have
hundreds of
thousands of origins
of replication.
DNA Replication
• Here is an electron
micrograph and a
schematic
representation of
bacterial DNA
replication.
DNA Replication
• Helicase attaches to origin of replication
and unzips DNA by breaking hydrogen
bonds
DNA Replication
• Primers are the short nucleotide fragments (DNA
or RNA) to which DNA polymerase will add
nucleotides according to the base paring rules.
• Primase is the enzyme that creates a primer that
can initiate the synthesis of a new DNA strand.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A772
Priming for DNA Synthesis
• Primase (an RNA
polymerase) adds RNA
primer to strand
DNA Replication
• DNA polymerases
are enzymes that
catalyze the
elongation of DNA
at the replication
fork.
• One by one,
nucleotides are
added by DNA
polymerase to the
growing end of the
DNA strand.
Elongating a New Strand
• DNA polymerases can only attach nucleotides to
the 3’-OH end of a growing daughter strand
• Thus, replication always proceeds in the 5’ to 3’
direction
• The strands in the double helix are antiparallel.
• The sugar-phosphate backbones run in opposite
directions.
– Each DNA strand has a 3’
end with a free hydroxyl
group attached to
deoxyribose and a 5’ end
with a free phosphate
group attached to
deoxyribose.
– The 5’ -> 3’ direction of
one strand runs counter to
the 3’ -> 5’ direction of
the other strand.
Fig. 16.12
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Strands of DNA are said to be
Complementary (Anti-Parallel)
• So, if one strand
is known, the
other strand can
be determined
3’ A = T 5’
C
G
G
T
A
T
C
C
5’
G
C
C
=A
=T
=A
G
G
3’
DNA Replication
• Another DNA
Polymerase
removes the RNA
primer and
replaces RNA
bases with
complementary
DNA bases
DNA Replication
• DNA ligase then
forms covalent
bonds between
DNA fragments
Uh-Oh…Problem
• Since DNA polymerases can only add
nucleotides to the free 3’ end of a growing DNA
strand.
– This creates a problem at the replication fork because
one parental strand is oriented 3’ → 5’ into the fork,
while the other antiparallel parental strand is oriented
5’ → 3’ into the fork.
• At the replication fork, one parental strand
(3’→ 5’ into the fork), the leading strand, can be
used by polymerases as a template for a
continuous complementary strand.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The other parental strand (5’ → 3’ into the
fork), the lagging strand, is copied away
from the fork in short segments (Okazaki
fragments).
• Okazaki fragments,
each about 100-200
nucleotides, are joined
by DNA ligase to form
the sugar-phosphate
backbone of a single
DNA strand.
Fig. 16.13
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
DNA Replication
Checking for Errors
• Mistakes during the initial pairing of template
nucleotides and complementary nucleotides
occur at a rate of one error per 10,000 base
pairs.
• DNA polymerases have a “proofreading” role
– Can only add nucleotide to a growing strand if
the previous nucleotide is correctly paired to
its complementary base
• If mistake happens, DNA polymerase backtracks,
removes the incorrect nucleotide, and replaces it
with the correct base
• The final error rate is only one per billion
nucleotides.
• DNA molecules are constantly subjected to
potentially harmful chemical and physical agents.
– Reactive chemicals, radioactive emissions, X-rays,
and ultraviolet light can change nucleotides in
ways that can affect encoded genetic information.
– DNA bases often undergo spontaneous chemical
changes under normal cellular conditions.
• Mismatched nucleotides that are missed by DNA
polymerase or mutations that occur after DNA
synthesis is completed can often be repaired.
– Each cell continually monitors and repairs its
genetic material, with over 130 repair enzymes
identified in humans.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In mismatch repair, special enzymes fix incorrectly
paired nucleotides.
– A hereditary defect in one of these enzymes is
associated with a form of colon cancer.
• In nucleotide excision
repair, a nuclease cuts
out a segment of a
damaged strand.
– The gap is filled in by
DNA polymerase and
ligase.
– Used by skin cells when
repairing genetic damage
caused by UV rays of
sunlight
Fig. 16.17
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The importance of the proper functioning of repair
enzymes is clear from the inherited disorder
xeroderma pigmentosum.
– These individuals are hypersensitive to sunlight.
– In particular, ultraviolet light can produce thymine
dimers between adjacent thymine nucleotides.
– This buckles the DNA double helix and interferes with
DNA replication.
– In individuals with this disorder, mutations in their skin
cells are left uncorrected and cause skin cancer.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
http://www.photobiology.com/photobiology2000/applegate/index_files/image002.gif
The ends of DNA molecules are
replicated by a special mechanism
• Limitations in the DNA polymerase create
problems for the linear DNA of eukaryotic
chromosomes.
• The usual replication machinery provides
no way to complete the 5’ ends of
daughter DNA strands.
– Repeated rounds of replication produce
shorter and shorter DNA molecules.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
EndReplication
Problems
• The ends of eukaryotic chromosomal DNA
molecules, the telomeres, have special
nucleotide sequences.
– In human telomeres, this sequence is typically
TTAGGG, repeated between 100 and 1,000
times.
• Telomeres protect genes from being
eroded through multiple rounds of DNA
replication.
Fig. 16.19a
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Eukaryotic cells have evolved a mechanism
to restore shortened telomeres.
• Telomerase uses a short molecule of RNA
as a template to extend the 3’ end of the
telomere.
– There is now room for
primase and DNA
polymerase to extend
the 5’ end.
– It does not repair the
3’-end “overhang,”
but it does lengthen
the telomere.
Fig. 16.19b
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings