DNA and the Molecular Basis of Heredity
The Search for the Basis of Heredity
- his group showed that genes are located on chromosomes
- two molecules became the top candidates for the molecule of heredity
- protein, the most likely candidate, since there were 20 amino acids
- DNA, the less likely candidate, since there are only 4 nucleotides
- a third theory, DNA + protein, was also considered
Avery, Maclyn McCarty, and Colin MacLeod (1944)
- British Medical Officer, served in WWI
- worked on Streptococcus pneumoniae
- Two strains of bacteria - Rough (R) and Smooth (S)
- Rough - bacteria had a rough appearance in culture,
non-virulent (doesn't kill)
- Smooth - bacteria had a smooth appearance in culture,
- Performed several experiments whose results can be summarized as follows:
- Mouse + Live S >> Died. Autopsy revealed infestation of S strain
- Mouse + Live R >> Lived a happy life
- These two experiments illustrate the effects of normal R and S
- Mouse + Heat-killed S >> Lived a happy life
- Mouse + Heat-killed R >> Lived a happy life
- These two experiments illustrate that dead bacteria by themselves
- Mouse + Heat-killed S + Live R >> Died. Autopsy revealed
infestation of S strain
- Apparently something from the heat-killed S changed the live R to make
them virulent - this was called transformation
- Griffith didn't know what it was that transformed the R strain
into the S strain, but he demonstrated that it could be done
Hershey and Martha Chase (1952)
- Performed the same experiment, only they used tissue cultures growing in
- Heat-killed S + Live R + protease >> Live R and S colonies
- Heat-killed S + Live R + DNAse >> No growth
- Technically, this does not prove DNA is the carrier of genetic
information, but it provided strong circumstantial evidence
- Other experiments - purified each chemical class associated with
transformation and subjected bacteria to each one individually
- Only DNA produced transformation
- Their results were met with much resistance - even if DNA was accepted as
the molecule of heredity, very little was know about it and no one could
possibly imagine how such a uniform molecule could possibly contain all of
the information necessary to make you and me!
- Worked with radiolabled T2 bacteriophages
- Bacteriophage - composed only of protein coat and DNA - injected
something inside host bacterial cell which contained its genetic
- Was the injected material DNA or protein?
Additional Evidence That DNA Is the Genetic Material of Cells
- In the first experiment, illustrated on the left, the "hot"
substance was the protein coat.
- In the second experiment, illustrated on the right, the "hot"
substance was the DNA.
- After the bacteriophage had injected the genetic material into the cell,
the solutions were centrifuged and each reaction flask was measured for
- In the first experiment, all of the radioactivity was in the fluid that
the cells were originally suspended.
- In the second experiment, all of the radioactivity was in the bacteria.
- It was concluded that since the radioactivity in the second experiment was
in the bacteria, the genetic information was contained in the DNA and not
James Watson and Francis Crick - The Double Helix Model of DNA
- Prior to mitosis, eukaryotic cells exactly double their DNA
- This DNA is distributed exactly between the two daughter cells
- Diploid cells have exactly twice as much DNA as the haploid gametes found
- Chargaff's Rule
- The DNA composition differs between organism to organism and species
- In ALL organisms, there are equal numbers of A and T. Likewise,
there are equal numbers of C and G.
- The A/G, A/C, T/G, T/C ratios, however, differ from person to person,
species to species
There was a race to elucidate the 3-D structure of DNA
The Watson-Crick Model of DNA
- Linus Pauling in California proposed a triple
- Maurice Wilkins and Rosalind
Franklin in London were also hot on the trail
- However, the first scientists to properly describe the structure of DNA
were two relatively unknown Americans, James
Watson and Francis Crick
- Read the actual paper here
- note, there ain't much there, is there?
- Read a Time Magazine blurb on the dynamic duo here
- More info on the people involved in this study here
- DNA is composed of Four Nucleotides, Adenine (A), Guanosine (aka Guanine,
G), Thymine (T) and Cytosine (C)
- Adenine and Guanine are Purines - they are larger and have a double
- Thymine and Cytosine are Pyrmidines - they are smaller and have a
- The nucleotides have three components, a pentose sugar, an amino base (the
A, T, C, G), and a phosphate group
- The deoxyribose sugar of the DNA backbone has 5 carbons and 3 oxygens.
- The carbon atoms are numbered 1', 2', 3', 4', and 5' to distinguish
from the numbering of the atoms of the purine and pyrmidine rings.
- The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate
groups to form the DNA backbone.
- Deoxyribose lacks an hydroxyl group at the 2'-position when compared
to ribose, the sugar component of RNA.
Structure of Deoxyribose
- The Nucleotides are arranged in an anti-parallel
double helix with Adenine pairing with Thymine and Guanine pairing
with Cytosine via hydrogen bonds
- A purine always pairs with a pyrmidine (A-T has two H-bonds, G-C has
- One strand runs 5' to 3' while the other runs 3' to 5'
DNA Replication - is DNA replication conservative, semiconservative, or
||Conservative - old strand acts as a template
- One daughter strand is the original template while the other
strand is composed entirely out of new nucleotides
Semiconservative - old strand splits apart and acts as a template
- Each strand of both daughter molecules contains a mixture of old
and newly synthesized DNA parts
Isotopes were used to prove that DNA replication was semiconservative
- Both daughter strands are composed of one of the old strands and
one comprised out of new nucleotides
Meselson - Stahl Experiment
- E. coli bacteria were cultured for several generations in heavy
nitrogen (15N - normal nitrogen is 14N)
- The bacteria incorporated 15N into their nucleotides and
thus, their DNA
- Meselson and Stahl then transferred the bacteria to a medium containing 14N,
- Thus, any DNA that the bacteria synthesized would be lighter than the
"old" DNA made with the 15N medium
- The DNA was extracted from the cells and centrifuged in a cesium chloride
density gradient for 20 hours at 40,000rpm.
- The DNA migrated to a point that was equivalent to their density.
- Results from the parental generation contained only a single high
density band - all DNA molecules contained the "heavy"
- DNA taken from the two generations after the switch contained an
intermediate-density band - DNA contained a "heavy" DNA strand
from the parent and a complementary "light" DNA strand.
- Density results from generation 3, displayed two bands. They included
an intermediate density band, composed of one parental "heavy"
strand and a new light band, composed of only DNA strands with
- Mixing the first and third generations showed all three types of DNA
molecules - heavy, light and intermediate.
- For a diagram of the experimental procedure, click here
- Self test: what were the predicted results of the experiment for
conservative, semiconservative, and dispersive
It is very important to know that DNA replication is not a passive and
spontaneous process. Many enzymes are required to unwind the double helix and to
synthesize a new strand of DNA. We will approach the study of the molecular
mechanism of DNA replication from the point of view of the machinery that is
required to accomplish it. The unwound helix, with each strand being synthesized
into a new double helix, is called the replication fork.
The Enzymes of DNA Replication
The Steps of DNA Replication
- Topoisomerase is responsible for eliminating supercoiling in DNA.
The tension holding the helix in its coiled and supercoiled structure can be
broken by nicking a single strand of DNA. Try this with string. Twist two
strings together, holding both the top and the bottom. If you cut only one
of the two strings, the tension of the twisting is released and the strings
- Helicase accomplishes unwinding of the original double strand, once
supercoiling has been eliminated by the topoisomerase. The two strands very
much want to bind together because of their hydrogen bonding affinity for
each other, so the helicase activity requires energy (in the form of ATP )
to break the strands apart.
- DNA polymerase proceeds along a single-stranded molecule of DNA,
recruiting free dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond
with their appropriate complementary dNTP on the single strand (A with T and
G with C), and to form a covalent phosphodiester bond with the previous
nucleotide of the same strand. The energy stored in the triphosphate is used
to covalently bind each new nucleotide to the growing second strand. There
are different forms of DNA polymerase , but it is DNA polymerase III that is
responsible for the processive synthesis of new DNA strands. DNA polymerase
cannot start synthesizing de novo on a bare single strand. It needs a
primer with a 3'OH group onto which it can attach a dNTP. DNA polymerase is
actually an aggregate of several different protein subunits, so it is often
called a holoenzyme. The holoenzyme also has proofreading activities, so
that it can make sure that it inserted the right base, and nuclease
(excision of nucleotides) activities so that it can cut away any mistakes it
might have made.
- Primase is actually part of an aggregate of proteins called the
primeosome. This enzyme attaches a small RNA primer to the single-stranded
DNA to act as a substitute 3'OH for DNA polymerase to begin synthesizing
from. This RNA primer is eventually removed by RNase H and the gap is filled
in by DNA polymerase I.
- Ligase can catalyze the formation of a phosphodiester bond given an
unattached but adjacent 3'OH and 5'phosphate. This can fill in the
unattached gap left when the RNA primer is removed and filled in. The DNA
polymerase can organize the bond on the 5' end of the primer, but ligase is
needed to make the bond on the 3' end.
- Single-stranded binding proteins are important to maintain the
stability of the replication fork. Single-stranded DNA is very labile, or
unstable, so these proteins bind to it while it remains single stranded and
keep it from being degraded.
- A portion of the double helix is unwound by a helicase.
- A molecule of DNA polymerase binds to one strand of the DNA and
begins moving along it in the 3' to 5' direction, using it as a
template for assembling a leading strand of nucleotides and
reforming a double helix.
- Because DNA synthesis can only occur 5' to 3', a second DNA
polymerase molecule is used to bind to the other template strand as
the double helix opens. This molecule must synthesize discontinuous
segments of polynucleotides (called Okazaki fragments). Another
enzyme, DNA ligase then stitches these together into the lagging
Speed of Replication
- Prokaryotes - The single molecule of DNA that is the E. coli genome
contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single,
fixed location in this molecule, the replication origin, proceeds at
about 1000 nucleotides per second, and thus is done in no more than 40
minutes. And thanks to the precision of the process (which includes a
"proof-reading" function), the job is done with only about one
incorrect nucleotide for every 109 nucleotides inserted. In other words,
more often than not, the E. coli genome (4.7 x 106) is copied without error!
- Eukaryotes - The average human chromosome contains 150 x 106
nucleotide pairs which are copied at about 50 base pairs per second. The
process would take a month (rather than the hour it actually does) but for
the fact that there are many replication origins on the eukaryotic
chromosome. Replication begins at some origins earlier in S phase than at
others, but the process is completed for all by the end of S phase. As
replication nears completion, "bubbles" of newly replicated DNA
meet and fuse, finally forming two new molecules
to see a shockwave graphic on DNA replication
Enzymes Proofread DNA During Its Replication and Repair Damage in Existing
We cannot attribute the accurate of DNA replication solely to the specificity
of base pairing
- Base pairing specificity has an error rate of one in 10,000 base pairs
- However, errors only occur in one in 1,000,000,000 base pairs (one in a
- Mismatch repair - during DNA replication, DNA polymerase proofreads
the DNA as soon as it is added to the strand searching for mismatches
- When found, the polymerase removes the mismatched nucleotide, adds a
correct nucleotide, and continues synthesizing DNA
- DNA is also constantly exposed to environmental hazards (UV, X-rays,
carcinogens, mutagens, etc.)
- Enzyme systems constantly moniter DNA looking for altered DNA
- Example - UV radiation causes two adjacent Thymines to form a Thymine
- When found, nuclase enzymes remove the TT dimer and a few
- DNA polymerase fills in the gap
- ligase seales the remaining nick
- Over 100 DNA repair enzyme are known in E. coli alone!
Telomeres - The DNA Synthesis Termination Paradox
For linear DNA (i.e. eukaryotic chromosomes) the fact that DNA polymerase can
only add nucleotides to the 3' end of a preexisting polynucleotide presents a
- Prokaryotes - they have circular DNA so this is not a problem
- Eukaryotes have a special DNA sequences called a telomeres on the
terminal regions of their chromosomes
- Telomeres do not contain genes; instead, they contain repeating units
of one short nucleotide sequence, usually TTAGGG
- Telomeric DNA protects the organism's genes from being eroded through
successive rounds of DNA replication
- Telomeric DNA aids in preventing separate chromosomes from fusing
- Telomeric DNA and associated proteins prevent the ends from activating
the cell's systems for monitoring DNA damage
- A DNA molecule detected as broken may trigger signal-transduction
pathways leading to cell-cycle arrest or death
- Telomerase - a special enzyme that catalyzes the lengthening of
- Telomerase is special in having a piece of RNA associated with its
- This RNA serves as a template so DNA Polymerase can extend the end of
- Since the telomeric units are repeating, this can occur many times