MOLECULAR & CELLULAR
NEUROBIOLOGY
Master Course Cognitive Neuroscience - Radboud
University, Nijmegen
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Chapter 2: Cells
and within cells |
More on DNA
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Understanding DNA: the molecule of life
We already saw
that the DNA molecule is a linear
polynucleotide consisting of repeating units of nucleotides; nucleotides are the building stones
of DNA. Each nucleotide
consists of one base, a deoxyribose sugar, and a phosphate molecule. There are
four bases: adenine, cytosine, guanine, and thymine. Adenine and guanine are
purines and cytosine and thymine are pyrimidine bases. Each base is bound to one
sugar and one phosphorous molecule. The ribose molecule lacks a hydroxyl group
(OH) at the number 2 carbon on the ring and thus the designation
deoxyribonucleic acid. The purine bases (adenine and quanine) are two-ringed
structures and the pyrimidine bases (thymidine and cytosine) are the
single-ringed structures (Fig 1).
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Figure
1. Building blocks of nucleic acids, DNA comprises a deoxyribose sugar backbone
with the nucleotide bases adenine, guanine, cytosine, and thymidine attached to
the C1 position carbon on the sugar ring. |
There are four different nucleotides :
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dATP: deoxyadenosine triphosphate
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dGTP: deoxyguanosine triphosphate
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dTTP: deoxythymidine triphosphate
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dCTP: deoxycytidine triphosphate
For convenience, these four nucleotides are called dNTPs (deoxynucleoside
triphosphates). A nucleotide is thus made of three major parts: a nitrogen-containing base,
a sugar molecule and a triphosphate. Only the nitrogen base is different in
the four nucleotides.
DNA is formed by coupling the nucleotides between the phosphate group from a
nucleotide (which is positioned on the 5th C-atom of the sugar
molecule) with the hydroxyl on the 3rd C-atom on the sugar molecule of
the previous nucleotide. To accomplish this, a diphosphate molecule is split
off (and releases energy). This means that new nucleotides are always added on
the 3' side of the chain.
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Structure of DNA
Utilizing X-ray diffraction
data, obtained from crystals of DNA, James Watson and Francis
Crick proposed a model for the structure of DNA. This model (subsequently
verified by additional data) predicted that DNA would exist as a
helix of two complementary antiparallel strands, wound around
each other in a rightward direction and stabilized by H-bonding
between bases in adjacent strands. In the Watson-Crick model,
the bases are in the interior of the helix aligned at a nearly
90 degree angle relative to the axis of the helix. Purine bases
form hydrogen bonds with pyrimidines, in the crucial phenomenon
of base pairing. Experimental determination has shown that, in
any given molecule of DNA, the concentration of adenine (A) is
equal to thymine (T) and the concentration of cytidine (C) is
equal to guanine (G). This means that A will only base-pair with
T, and C with G. According to this pattern, known as
Watson-Crick base-pairing, .
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A-T
Base Pair
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G-C
Base Pair
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The double-helix structure
thus arises from the complementary pairing between the purine
base of one strand and the pyrimidine base of the second strand. The
complementary pairing is optimally stabilized by the hydrogen bonds formed between
the purine and the pyrimidine base, such that guanine (G) will always pair with
cytosine (C) via three hydrogen bonds and adenine (A) will always pair with
thymidine (T) via two hydrogen bonds (Fig 2). The base-pairs composed of
G and C thus contain three H-bonds, whereas those of A and T
contain two H-bonds. This makes G-C base-pairs more stable than
A-T base-pairs. Because of incompatible
ring conformations, cross pairing between the other purines and pyrimidines, ie,
adenine with guanine and cytosine with thymine, do not occur. This complementary
base pairing is the basis of molecular genetics. The binding of a phosphorus
molecule to each nucleoside gives rise to a nucleotide. When each nucleotide
strand is considered individually, the sequence of nucleotide bases is always
read from left to right from the 5′ carbon to 3′ carbon direction (Fig 3).
In the double-helix configuration, the strand
complementary to the 5′ to 3′ strand is oriented in a 3′ to 5′ direction
relative to its complementary strand. This organization is referred to as the
antiparallel or antisense arrangement of the DNA strands. For example,
nucleotide pairing between a nucleotide strand with a 5′ ATCCG 3′ sequence has
as its complementary strand 3′ TAGGC 5′ in the double-helix configuration.
Additionally, when describing segments of DNA, it is common to refer to them by
size. For example, a segment of double-stranded DNA composed of 200 nucleotides
is often referred to as a 200-basepair (bp) segment. Similarly, 1000- and
1,000,000-nucleotide segments are 1-kb or 1000-kbp [1-megabase (Mb)] segments.
The antiparallel nature of
the helix thus stems from the orientation of the individual
strands: from any fixed position in the helix, one strand is
oriented in the 5'—>3' direction and the other in the 3'—>5'
direction. On its exterior surface, the double helix of DNA
contains two deep grooves between the ribose-phosphate chains.
These two grooves are of unequal size and termed the major and
minor grooves. The difference in their size is due to the
asymmetry of the deoxyribose rings and the structurally distinct
nature of the upper surface of a base-pair relative to the
bottom surface.
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Figue
2. Specificity of DNA basepairing. The two strands of DNA are bound together via
hydrogen bonds between the nucleotide bases on each strand. The bonds are formed
by strict pairing between two complementary bases, A T or C G, such
that each strand reflects the exact sequence of the opposite strand (A,
adenine; T, thymine; G, guanine; C, cytosine). The sugar
(dart pentamer) and phosphate (dark circle) linkages form the backbone of the
DNA strand. |
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Figure 3. DNA replication conserves the nucleotide sequence. DNA is a double-stranded helical
molecule bound together by the nucleotide bases contained on each individual
strand. During cell division, two identical copies of the original parental
strand are made by unwinding the DNA and then synthesizing a complementary
second strand to make two identical new daughter stands. |
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The double helix of DNA has
been shown to exist in several different forms, depending upon
sequence content and ionic conditions of crystal preparation.
The B-form of DNA prevails under physiological conditions of low
ionic strength and a high degree of hydration. Regions of the
helix that are rich in pCpG dinucleotides can exist in a novel
left-handed helical conformation termed Z-DNA. This conformation
results from a 180 degree change in the orientation of the bases
relative to that of the more common A- and B-DNA.
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Structure of B-DNA
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Structure of Z-DNA
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Thermal properties of DNA
As cells divide it is a
necessity that the DNA be copied (replicated), in such a way
that each daughter cell acquires the same amount of genetic
material. In order for this process to proceed the two strands
of the helix must first be separated, in a process termed
denaturation. This process can also be carried out in vitro. If
a solution of DNA is subjected to high temperature, the H-bonds
between bases become unstable and the strands of the helix
separate in a process of thermal denaturation. The base
composition of DNA varies widely from molecule to molecule and
even within different regions of the same molecule. Regions of
the duplex that have predominantly A-T base-pairs will be less
thermally stable than those rich in G-C base-pairs. In the
process of thermal denaturation, a point is reached at which 50%
of the DNA molecule exists as single strands. This point is the
melting temperature (Tm), and is characteristic of
the base composition of that DNA molecule. The Tm
depends upon several factors in addition to the base composition.
These include the chemical nature of the solvent and the
identities and concentrations of ions in the solution. When
thermally melted DNA is cooled, the complementary strands will
again re-form the correct base pairs, in a process is termed
annealing or hybridization. The rate of annealing is dependent
upon the nucleotide sequence of the two strands of DNA.
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The
genome
The genome refers to the
complete DNA sequence of an organism, which is enclosed in the nucleus of a cell
(Table 1). In the human, there are 3 billion
base pairs, which contain information that would more than fill a 500,000-page
textbook. It is estimated that, in a single individual, if all of the
chromosomes were joined end to end, it would reach from the earth to the moon
about 8000 times. The actual length of the DNA from each cell is not apparent
because the DNA helix of each chromosome exists as a compact, coiled structure
stabilized by protein molecules, many of which are histones. The compact nature
results in an increase in diameter, thus allowing the DNA to be visible by
electron microscopy. The coiled, compact character of DNA enables all of the
genetic information from a single cell to fit neatly into the cell’s nucleus,
which occupies less than 10% of the total cell volume. The ~21,000 genes encoding
a human being accounts for about 1% of the DNA, thus most of the DNA is
nonprotein coding, but 80% of the human genome is thought to be functional. There are 46 chromosomes and each chromosome is a long continuous
DNA molecule. The chromosomes vary in size, but even chromosome 21, the smallest
of them, contains more than 50,000,000 base pairs.
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Table 1. The human genome (completed April 2003)
Number of bases |
3.2
billion |
Genes
estimated |
~21,000 |
DNA
for genes |
~1.5% |
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