Intro.

to Nucleic Acid Chemistry

The role of DNA as a repository of genetic information depends in
part on its inherent stability.
The chemical transformations that occur to DNA are generally
very slow in the absence of an enzyme catalyst. However, even
very slow reactions that alter DNA structure are physiologically
significant.
Processes such as carcinogenesis and aging are intimately linked to
slowly accumulating, irreversible alterations of DNA. Other
nondestructive alterations such as strand separation prior to DNA
replication and transcription are essential to function. The
chemical behavior of DNA is the focus of the next several slides.
DNA was first isolated and characterized by Friedrich Miescher in 1868. He called the phosphorus-
containing substance “nuclein.” Not until the 1940s, with the work of Oswald T. Avery, Colin MacLeod,
and Maclyn McCarty, was there any compelling evidence that DNA was the genetic material.
Avery and his colleagues found that DNA extracted from a virulent (disease-causing) strain of the
bacterium Streptococcus pneumoniae and injected into a nonvirulent strain of the same bacterium
transformed the nonvirulent strain into a virulent strain. They concluded that the DNA from the virulent
strain carried the genetic information for virulence
1952, experiments by
Alfred D. Hershey and
Martha
Chase, in which they
studied the infection of
bacterial
cells by a virus
(bacteriophage) with
radioactively
labeled DNA or protein,
removed any remaining
doubt
that DNA, not protein,
carried the genetic
information.
Another important clue
to the structure of DNA
 Chargaff’s rules
Another important clue to the structure of DNA came from the work of Erwin Chargaff
and his colleagues in the late 1940s. They found that the four nucleotide bases of DNA
occur in different ratios in the DNAs of different organisms and that the amounts of
certain bases are closely related. These data, collected from DNAs of a great many
different species, led Chargaff to the following conclusions:
1. The base composition of DNA generally varies from one species to another.

2. DNA specimens isolated from different tissues of the same species have the same base
composition.

3. The base composition of DNA in a given species does not change with an organism’s
age, nutritional state, or changing environment.

4. In all cellular DNAs, regardless of the species, the number of adenosine residues is
equal to the number of thymidine residues (that is, A = T), and the number of guanosine
residues is equal to the number of cytidine residues (G = C).

From these relationships it follows that the sum of the purine residues equals the sum of
the pyrimidine residues; that is, A + G = T + C. These quantitative relationships,
sometimes called “Chargaff’s rules,”
The complementary antiparallel
strands of DNA follow the pairing
rules proposed by Watson and Crick.
The base-paired antiparallel strands
differ in base composition: the left
strand has the composition
A3T2G1C3; the right, A2T3G3C1.
They also differ in sequence when
each chain is read in the 5’ 3’
direction. Note the base equivalences:
A = T and G = C in the duplex.
In samples of DNA isolated from two unidentified species
of bacteria, X and Y, adenine makes up 32% and
17%, respectively, of the total bases. What relative
proportions of adenine, guanine, thymine, and cytosine
would you expect to find in the two DNA samples? What
assumptions have you made? One of these species was
isolated from a hot spring (64 0C). Which species is most
likely the thermophilic bacterium, and why?
Hoogsteen pairing
 Denaturation and Annealing of DNA
Double-helical DNA can be denatured (melted) to single-
stranded DNA by heating and extremes of pH.
Disruption of the hydrogen bonds between paired bases and
of base stacking causes unwinding of the double helix to
form two single strands, completely separate from each
other along the entire length or part of the length (partial
denaturation) of the molecule (Fig. 8-26).
Covalent bonds in the DNA are not broken by denaturation.
When the temperature or pH is returned to the range in
which most organisms live, the unwound segments of the
two strands spontaneously rewind, or anneal, to yield the
intact double helix.
The renaturation of completely melted DNA occurs in two
steps. First, the two strands slowly find each other by
random collisions and form a short segment of
complementary double helix.
Second, the remaining unpaired bases rapidly zipper
themselves together to form the complete double helix.
The melting of double-helical DNA can be followed by
measuring the increase in absorption of UV light (260 nm)
on melting (the hyperchromic effect).
 Heat Denaturation of DNA
Every species of double-helical DNA has a characteristic
denaturation temperature, or melting point (tm ; formally
the temperature at which half of the DNA is present as
separated single strands) (Fig. 8-27).
The melting point is dependent on, and rises with, the
content of G/C base pairs in the DNA.
This is because G/C base pairs are held together more
tightly, by three hydrogen bonds, than are A/T pairs (two
hydrogen bonds).
The energetic requirements for DNA melting explain why
DNA at replication origins, and at promoters used in gene
transcription is enriched in A/T base pairs.

RNA-RNA double helices and DNA-RNA hybrid double

helices melt at higher temperatures than double-helical
DNAs of comparable base composition, for unknown
reasons.
 DNA Hybridization
The ability of two complementary DNA strands to pair (hybridize) with one another can be
used to detect similar DNA sequences in two different species or within the genome of a
single species (Fig. 8-29).
To perform these analyses, the DNA samples to be compared are first completely denatured
by heating. The solutions then are mixed and slowly cooled. Some DNA strands of each
sample associate with their normal complementary partners and anneal to form duplexes.

If the two DNAs have significant sequence similarity, they also tend to form partial duplexes
or hybrids with each other.

The greater the sequence

similarity between the two DNAs,
the greater the number of hybrids
formed.
The extent of hybrid formation
reflects how closely related the
organisms being analyzed are to
one another.
For example, human DNA
hybridizes much more extensively
with mouse DNA than with yeast
DNA. Hybridization techniques are
commonly used in many modern
 Deamination of Nucleotides in DNA
Purines and pyrimidines, along with the nucleotides
of which they are a part, undergo spontaneous
alterations in their covalent structure which can
produce permanent changes (mutations) in the
genetic information.
One such modification is the spontaneous loss of
the exocyclic amino groups (deamination) present
in the bases of DNA (Fig. 8-30a).
For example, deamination of cytosine in DNA to
uracil occurs in about one of every 107 cytidine
residues in 24 hours under cellular conditions.
This corresponds to about 100 spontaneous events
per day in a mammalian cell. This reaction likely
explains why DNA contains thymine rather than
uracil.
Namely, uracils produced by cytosine deamination
can be specifically recognized and repaired back
to cytosine residues by enzymatic repair systems.
Without repair, cytosine deamination would
convert many G/C base pairs in DNA to A/U base
pairs.
 Depurination of Nucleotides in DNA
Another important reaction in DNA is
the hydrolysis of the N-ß-glycosyl bond
between the base and the pentose, to
create a DNA lesion called an AP
(apurinic, apyrimidinic) site or abasic
site (Fig. 8-30b). This reaction occurs
at a higher rate for purines than for
pyrimidines, and in the test tube is
accelerated in the presence of dilute
acid (pH 3). It is calculated that on
the order of one in 10 5 purines
(~10,000 per mammalian cell) are lost
from DNA daily under cellular conditions.
Again, repair systems must operate to
repair abasic sites in DNA to prevent
the accumulation of mutations.
 Formation of Pyrimidine Dimers in DNA
DNA also can be damaged by various
forms of UV and ionizing radiation. For
example, in the presence of near-UV
light (200 to 400 nm), adjacent
pyrimidine bases in nucleic acids
combine via their rings to form
cyclobutane pyrimidine dimers, and so-
called 6-4 photoproduct pyrimidine
dimers (Fig. 8-31a). Formation of a
cyclobutane pyrimidine dimer introduces
a bend or kink into the DNA (Fig.
8-31b). Pyrimidine dimers must be
removed from the template strand for
DNA replication to proceed normally.
Higher-energy ionizing radiation, (x
rays and gamma rays) can cause ring
opening and fragmentation of bases as
well as breaks in the covalent backbone
of DNA. It is estimated that UV and
ionizing radiations are responsible for
about 10% of all DNA damage caused
by environmental agents.
 DNA-damaging Chemical Agents (I)
DNA can be damaged by reactive chemicals introduced into the
environment as products of industrial activity. Agents that result
in deamination of bases are shown in Fig. 8-32a. All of these
agents are precursors of nitrous acid (HNO2), which is the
compound that actually is responsible for deamination. Bisulfate is
also a deamination agent. Some of these chemicals are used in
small amounts for food preservation.
 DNA-damaging Chemical Agents (II)
A broad class of chemicals that act as alkylating agents also
cause a significant amount of damage to the bases of DNA (Fig.
8-32b). For example, dimethylsulfate ((CH3)2SO4) can methylate
guanine to produce O6-methylguanine which can no longer base
pair with cytosine. The compound S-adenosylmethionine is a
cofactor used in enzymatic methylation of DNA. DNA methylation
is important in bacterial restriction-modification systems and in
mismatch repair of erroneously incorporated bases during
replication. Probably the most important source of mutagenic
alterations in DNA is oxidative damage.
 Sanger DNA Sequencing (I)
The Sanger method of DNA sequencing makes use of the
mechanism of DNA synthesis by DNA polymerases (Fig. 8-33a).
DNA polymerases require both a primer (a short oligonucleotide
strand), to which nucleotides are added, and a template strand to
guide the selection of each added nucleotide. The 3’-hydroxyl
group of the primer reacts with an incoming deoxynucleoside
triphosphate (dNTP) to form a new phosphodiester bond as the
chain grows in the 5’ to 3’ direction.
 Sanger DNA Sequencing (II)
The Sanger method uses dideoxynucleoside triphosphate (ddNTP)
analogs (Fig. 8-33b) to interrupt DNA synthesis. (The Sanger
method is also known as the dideoxy or chain-termination
method). When a ddNTP is inserted in place of a dNTP, strand
elongation is halted after the analog is added, because the
analog lacks the 3’-hydroxyl group needed for the addition of the
next nucleotide. An overview of the steps performed in Sanger
sequencing is presented in the next two slides.
 Sanger DNA Sequencing (III)
The DNA to be sequenced is used
as the template strand, and a
short oligonucleotide primer,
radioactively or fluorescently
labeled, is annealed to it (Fig.
8-33c). By addition of small
amounts of a single ddNTP, for
example, ddCTP, to an otherwise
normal reaction system, the
synthesized strands will be
prematurely terminated at some
locations where dC normally
occurs. Given the excess of dCTP
over ddCTP, the chance that the
analog will be incorporated
whenever a dC is to be added is
small. However, ddCTP is present
in sufficient amounts to ensure
that each new strand has a high
probability of acquiring a least
one ddC at some point during
synthesis. (Continued on the next
slide).
 Sanger DNA Sequencing (IV)
The result is a solution
containing a mixture of labeled
fragments, each ending with a
C residue. Each C residue in
the sequence generates a set
of fragments of a particular
length, such that the different-
sized fragments, separated by
electrophoresis, reveal the
location of C residues. This
procedure is repeated
separately for each of the four
ddNTPs, and the sequence can
be read directly from an
autoradiogram of the gel.
Because shorter DNA
fragments migrate faster, the
fragments located near the
bottom of the gel represent
the nucleotide positions closest
to the primer (the 5’ end), and
the sequence is read (in the 5’
to 3’ direction) from bottom to
top. Note that the sequence
obtained is that of the strand
complementary to the strand
being analyzed.
 Sanger DNA Sequencing (V)
Several high-throughput and automated
sequencing methods, based on the Sanger
method, are now used for rapid sequencing
of large segments of DNA. One such
method is illustrated in Fig. 8-34. In this
approach, each of the four
dideoxynucleotides used in chain-
termination is labeled with a different
fluorescent dye that gives all the
fragments terminating in that nucleotide a
particular color. All four labeled ddNTPs
are added to a single reaction tube. The
resulting dye-labeled segments of DNA
copied from the template are applied to a
single capillary gel and are subjected to
electrophoresis. The DNA sequence is read
by determining the sequence of colors in
the peaks as they pass through a laser
detector. Even more efficient methods for
high-throughput sequencing are discussed
in Chap. 9.
 Nucleoside Mono-, Di-, & Triphosphates
The 5’ hydroxyl group of a nucleotide commonly may have one, two,
or three phosphate groups attached to it. The resulting molecules
are referred to as nucleoside mono-, di-, and triphosphates (Fig.
8-36). Starting from the sugar ring, the phosphates are labeled α,
ß, and γ. As discussed in the next slide, the hydrolysis of
nucleoside triphosphates (particularly ATP) provides chemical
energy needed to drive many cellular reactions. Nucleoside
triphosphates also serve as the activated precursors of DNA and
RNA synthesis.
 ATP as a Source of Chemical Energy
ATP is the nucleotide that is most
commonly used as a source of
energy for biological processes. The
energy released by the hydrolysis
of ATP (and the other nucleoside
triphosphates) is accounted for by
the structure of the triphosphate
group. The bonds between the α-ß
and ß-γ phosphates of ATP are
phosphoanhydride linkages. The
hydrolysis of either of these bonds
liberates about 30 kJ/mol under
standard biochemical conditions (Fig.
8-37). When chemically coupled to
an energy-requiring (endergonic)
process, the hydrolysis of
phosphoanhydride bonds often
provides enough energy to drive the
process forward. In contrast, the
hydrolysis of the phosphoester
linkage between the ribose and the
α phosphate of ATP is less
exergonic, liberating about 14 kJ/
mol.
 Adenosine-containing Coenzymes
A variety of enzyme
cofactors serving a wide
range of chemical functions
contain adenosine (red
shading) as part of their
structure (Fig. 8-38). They
are unrelated structurally
except for the presence of
adenosine, and in none of
these cofactors does the
adenosine moiety participate
directly in the coenzyme
function. Instead, it is
recognized by the enzyme as
an important “handle” in the
binding of the coenzyme to
the enzyme. The coenzymes
shown in Fig. 8-38 play very
important roles in metabolism.
Coenzyme A functions in acyl
group transfer reactions.
NAD + and FAD function in
oxidation-reduction reactions.
 Regulatory Nucleotides
Hormonal signal transduction
systems often rely on a
nucleotide for intracellular
signal transmission. These
compounds (typically called
second messengers) are formed
by the binding of the hormone
to a cell surface receptor, and
cause changes in the activities
of intracellular proteins and
enzymes leading to the cellular
response. Two common second
messengers (cAMP and cGMP)
are shown in Fig. 8-39. For
example, cAMP plays a major
role in epinephrine control of
glycogen metabolism in the liver
and skeletal muscle.