Lehninger Principles of Biochemistry

1
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.
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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.
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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).
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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.
5
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).
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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.
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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 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.
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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 ?, ß, 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.
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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 ?-ß and ß-? 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.
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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.
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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.