Chap.

8B Nucleotides and Nucleic Acids

Some Basics
Nucleic Acid Structure
Nucleic Acid Chemistry
Other Functions of Nucleotides

Fig. 8-12. X-ray diffraction pattern of DNA

fibers.
 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.
 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
molecular biology procedures. (See
Chap. 9).
 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--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 105 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 ,
, and . 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 -
and - 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
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.