The Structure of DNA

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DNA 4Y - The structure of DNA

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- DNA is an information storage medium
- stores 'genetic information' in strings of four 'characters' (A, C, G, T)
- The information on DNA must be:

- read (transcription+translation)
- copied (replication)archived (chromatin etc.)
What is DNA for? - maintained/repaired

'edited' (recombination)
DNA- Deoxyribonucleic acid
it is deoxy because - which contains one less oxygen-containing hydroxyl group
RNA can be an enzyme unlike DNA
linear code of strings of info of those 4 characters
Watson and Crick discover that DNA structure is a double helix (Watson won nobel prize but had
to resign later for crude comments about african inferiority)
The Swiss biochemist Frederich Miescher first observed DNA in the late 1800s. But nearly a
century passed from that discovery until researchers unraveled the structure of the DNA
molecule and realized its central importance to biology.For many years, scientists debated which
DNA in 1953 molecule carried life's biological instructions. Most thought that DNA was too simple a molecule
to play such a critical role. Instead, they argued that proteins were more likely to carry out this
vital function because of their greater complexity and wider variety of forms.The importance of
DNA became clear in 1953 thanks to the work of James Watson*, Francis Crick, Maurice Wilkins
and Rosalind Franklin. By studying X-ray diffraction patterns and building models, the scientists
figured out the double helix structure of DNA - a structure that enables it to carry biological
information from one generation to the next.

The molecule now known as DNA was first identified in the 1860s by a Swiss chemist called
Johann Friedrich Miescher. Johann set out to research the key components of white blood cells?,
part of our body's immune system. The main source of these cells? was pus-coated bandages
collected from a nearby medical clinic.
Johann carried out experiments using salt solutions to understand more about what makes up
white blood cells. He noticed that, when he added acid to a solution of the cells, a substance
separated from the solution. This substance then dissolved again when an alkali was added. When
investigating this substance he realised that it had unexpected properties different to those of the
other proteins? he was familiar with. Johann called this mysterious substance 'nuclein', because he
believed it had come from the cell nucleus?. Unbeknown to him, Johann had discovered the
molecular basis of all life - DNA. He then set about finding ways to extract it in its pure form.
discovery of DNA began in 1860's
Johann was convinced of the importance of nuclein and came very close to uncovering its elusive
role, despite the simple tools and methods available to him. However, he lacked the skills to
communicate and promote what he had found to the wider scientific community. Ever the
perfectionist, he hesitated for long periods of time between experiments before he published his
results in 1874. Before then he primarily discussed his findings in private letters to his friends. As a
result, it was many decades before Johann Friedrich Miescher's discovery was fully appreciated
by the scientific community.
For many years, scientists continued to believe that proteins were the molecules that held all of
our genetic material. They believed that nuclein simply wasn't complex enough to contain all of
the information needed to make up a genome. Surely, one type of molecule could not account
for all the variation seen within species
DNA is made of chemical building blocks called nucleotides. These building blocks are made of
three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. To form a
strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups
alternating.The four types of nitrogen bases found in nucleotides are: adenine (A), thymine (T),
what is DNA made of guanine (G) and cytosine (C). The order, or sequence, of these bases determines what biological
instructions are contained in a strand of DNA. For example, the sequence ATCGTT might instruct
for blue eyes, while ATCGCT might instruct for brown.The complete DNA instruction book, or
genome, for a human contains about 3 billion bases and about 20,000 genes on 23 pairs of
chromosomes.
DNA contains the instructions needed for an organism to develop, survive and reproduce. To
carry out these functions, DNA sequences must be converted into messages that can be used to
produce proteins, which are the complex molecules that do most of the work in our bodies.Each
What does DNA do? DNA sequence that contains instructions to make a protein is known as a gene. The size of a gene
may vary greatly, ranging from about 1,000 bases to 1 million bases in humans. Genes only make
up about 1 percent of the DNA sequence. DNA sequences outside this 1 percent are involved in
DNA 4Y - The structure of DNAwhen, how and how much of a protein is made.
regulating
DNA's instructions are used to make proteins in a two-step process. First, enzymes read the
information in a DNA molecule and transcribe it into an intermediary molecule called messenger
ribonucleic acid, or mRNA.Next, the information contained in the mRNA molecule is translated
How are DNA sequences used to make proteins? into the "language" of amino acids, which are the building blocks of proteins. This language tells
the cell's protein-making machinery the precise order in which to link the amino acids to produce
a specific protein. This is a major task because there are 20 types of amino acids, which can be
placed in many different orders to form a wide variety of proteins
Scientist use the term "double helix" to describe DNA's winding, two-stranded chemical structure.
This shape - which looks much like a twisted ladder - gives DNA the power to pass along
biological instructions with great precision.To understand DNA's double helix from a chemical
standpoint, picture the sides of the ladder as strands of alternating sugar and phosphate groups -
strands that run in opposite directions. Each "rung" of the ladder is made up of two nitrogen
bases, paired together by hydrogen bonds. Because of the highly specific nature of this type of
chemical pairing, base A always pairs with base T, and likewise C with G. So, if you know the
sequence of the bases on one strand of a DNA double helix, it is a simple matter to figure out the
What is the DNA double helix? sequence of bases on the other strand.DNA's unique structure enables the molecule to copy
itself during cell division. When a cell prepares to divide, the DNA helix splits down the middle
and becomes two single strands. These single strands serve as templates for building two new,
double-stranded DNA molecules - each a replica of the original DNA molecule. In this process,
an A base is added wherever there is a T, a C where there is a G, and so on until all of the bases
once again have partners.
In addition, when proteins are being made, the double helix unwinds to allow a single strand of
DNA to serve as a template. This template strand is then transcribed into mRNA, which is a
molecule that conveys vital instructions to the cell's protein-making machinery.

DNA is a long polymer made from repeating units called nucleotides, each of which is usually
symbolized by a single letter: either A, T, C, or G.[7][8] The structure of DNA is dynamic along its
length, being capable of coiling into tight loops and other shapes.[9] In all species it is composed
of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the
same axis, and have the same pitch of 34 angstroms (Å) (3.4 nanometres). The pair of chains has a
radius of 10 angstroms (1.0 nanometre).[10] According to another study, when measured in a
different solution, the DNA chain measured 22 to 26 angstroms wide (2.2 to 2.6 nanometres), and
one nucleotide unit measured 3.3 Å (0.33 nm) long.[11] Although each individual nucleotide is
very small, a DNA polymer can be very large and contain hundreds of millions, such as in
chromosome 1. Chromosome 1 is the largest human chromosome with approximately 220 million
base pairs, and would be 85 mm long if straightened.[12]
DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly
together.[10][13] These two long strands coil around each other, in the shape of a double helix.
The nucleotide contains both a segment of the backbone of the molecule (which holds the chain
together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase
linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more
phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as
in DNA) is called a polynucleotide.[14]
The backbone of the DNA strand is made from alternating phosphate and sugar groups.[15] The
sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined
DNA structure
together by phosphate groups that form phosphodiester bonds between the third and fifth
carbon atoms of adjacent sugar rings. These are known as the 3′-end(three prime end), and 5′-
end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms
from those of the base to which the deoxyribose forms a glycosidic bond. Therefore, any DNA
strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a
ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to
the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the
sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA
strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to
their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA
strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′
end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major
difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced
by the alternative pentose sugar ribose in RNA
The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides
and base-stacking interactions among aromatic nucleobases. The four bases found in DNA are
adenine (A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the
sugar-phosphate to form the complete nucleotide, as shown for adenosine monophosphate.
Adenine pairs with thymine and guanine pairs with cytosine, forming A-T and G-C base pairs
The DNA double helix is made of two antiparallel, base-
paired strands. Each strand has the structure shown here
"The uniqueness of a given DNA structure lies solely in the

sequence of its bases."
each of these strands has the chemical structure// each

unit looks exactly the same except for the base ( can be
The building blocks of DNA
either C,A,T,G) // no base ( backbone which is sugar and
phosphate )
- millions of nucleotides
sequence is bases is what's unique
Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
The building blocks of DNA

purines/pyrimidines
- bases are flat ( double bonded )
numbering of bases = purines

Nucleotides containing 2-deoxy-D-ribose as the pentose
component.
A 'chemist's' drawing of a nucleotide, showing the

conventional numbering of the deoxyribose, and where
the 5' and 3' phosphates are attached in DNA
- oxygen
Deoxyribonucleotides
- deoxyribose contains 5 carbon atoms
to distinguish the atoms from the base, there's a priming

numbering system
5'C - not part of the ring that's attached to a phosphate
3'c - important positions / chain carries on from this point

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms,
although, only B-DNA and Z-DNA have been directly observed in functional organisms.[15] The
conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and
direction of supercoiling, chemical modifications of the bases, the type and concentration of
metal ions, and the presence of polyamines in solution.[41]
The first published reports of A-DNA X-ray diffraction patterns—and also B-DNA—used analyses
based on Patterson transforms that provided only a limited amount of structural information for
oriented fibers of DNA.[42][43] An alternative analysis was then proposed by Wilkins et al., in
1953, for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in
terms of squares of Bessel functions.[44] In the same journal, James Watson and Francis
Crickpresented their molecular modeling analysis of the DNA X-ray diffraction patterns to
suggest that the structure was a double-helix.[10]
Although the B-DNA form is most common under the conditions found in cells,[45] it is not a
well-defined conformation but a family of related DNA conformations[46] that occur at the high
hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are
Conformations of the deoxyribose 5-membered
characteristic of molecular paracrystals with a significant degree of disorder.[47][48]
ring
Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor
groove and a narrower, deeper major groove. The A form occurs under non-physiological
conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid
pairings of DNA and RNA strands, and in enzyme-DNA complexes.[49][50] Segments of DNA
where the bases have been chemically modified by methylation may undergo a larger change in
conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed
spiral, the opposite of the more common B form.[51] These unusual structures can be recognized
by specific Z-DNA binding proteins and may be involved in the regulation of transcription.[52] A
2020 study concluded that DNA turned right-handed due to ionization by cosmic rays.
Three major forms of DNA are double stranded and connected by interactions between
complementary base pairs. These are terms A-form, B-form,and Z-form DNA

The information from the base composition of DNA, the knowledge of dinucleotide structure,
and the insight that the X‑ray crystallography suggested a helical periodicity were combined by
Watson and Crick in 1953 in their proposed model for a double helical structure for DNA. They
proposed two strands of DNA -- each in a right‑hand helix -- wound around the same axis. The
two strands are held together by H‑bonding between the bases (in anti conformation).Bases fit in
the double helical model if pyrimidine on one strand is always paired with purine on the other.
From Chargaff's rules, the two strands will pair A with T and G with C. This pairs a keto base with
an amino base, a purine with a pyrimidine. Two H‑bonds can form between A and T, and three can
form between G and C. This third H-bond in the G:C base pair is between the additional exocyclic
B-form DNA
amino group on G and the C2 keto group on C. The pyrimidine C2 keto group is not involved in
hydrogen bonding in the A:T base pair.
These are the complementary base pairs. The base‑pairing scheme immediately suggests a way to
replicate and copy the the genetic information.The two strands are not in a simple side‑by‑side
arrangement, which would be called a paranemic joint (Figure 2.5.32.5.3). (This will be
encountered during recombination in Chapter 8.) Rather the two strands are coiled around the
same helical axis and are intertwined with themselves (which is referred to as a plectonemic coil).
One consequence of this intertwining is that the two strands cannot be separated without the
DNA rotating, one turn of the DNA for every "untwisting" of the two strands
The DNA forms 2 grooves. most proteins bind to the major groove.
The major groove occurs where the backbones are far apart, the minor groove occurs where
they are close together.
The major groove is wider than the minor groove in DNA , and many sequence specific proteins
Major and minor groove
interact in the major groove. The N7 and C6 groups of purines and the C4 and C5 groups of
pyrimidines face into the major groove, thus they can make specific contacts with amino acids in
DNA-binding proteins. Thus specific amino acids serve as H‑bond donors and acceptors to form
H-bonds with specific nucleotides in the DNA. H‑bond donors and acceptors are also in the
minor groove, and indeed some proteins bind specifically in the minor groove. Base pairs stack,
with some rotation between them

Three different forms of duplex nucleic acid have been described. The most common form,
present in most DNA at neutral pH and physiological salt concentrations, is B-form. That is the
classic, right-handed double helical structure we have been discussing. A thicker right-handed
duplex with a shorter distance between the base pairs has been described for RNA-DNA
duplexes and RNA-RNA duplexes. This is called A-form nucleic acid.
A‑form nucleic acids and Z‑DNA A third form of duplex DNA has a strikingly different, left-handed helical structure. This Z DNA is
formed by stretches of alternating purines and pyrimidines, e.g. GCGCGC, especially in
negatively supercoiled DNA. A small amount of the DNA in a cell exists in the Z form. It has been
tantalizing to propose that this different structure is involved in some way in regulation of some
cellular function, such as transcription or regulation, but conclusive evidence for or against this
proposal is not available yet
The major difference between A-form and B-form nucleic acid is in the conformation of the
deoxyribose sugar ring. It is in the C2' endoconformation for B-form, whereas it is in the C3'
endoconformation in A-form. , if you consider the plane defined by the C4'-O-C1' atoms of the
deoxyribose, in the C2' endoconformation, the C2' atom is above the plane, whereas the C3'
atom is above the plane in the C3' endoconformation. The latter conformation brings the 5' and 3'
Differences between A-form and B-form nucleic hydroxyls (both esterified to the phosphates linking to the next nucleotides) closer together than
acid is seen in the C2' endoconfromation . Thus the distance between adjacent nucleotides is reduced
by about 1 Angstrom in A-form relative to B-form nucleic acid. A second major difference
between A-form and B-form nucleic acid is the placement of base-pairs within the duplex. In B-
form, the base-pairs are almost centered over the helical axis , but in A-form, they are displaced
away from the central axis and closer to the major groove. The result is a ribbon-like helix with a
more open cylindrical core in A-form.
Z-DNA is a radically different duplex structure, with the two strands coiling in left-handed helices
and a pronounced zig-zag (hence the name) pattern in the phosphodiester backbone. As
previously mentioned, Z-DNA can form when the DNA is in an alternating purine-pyrimidine
sequence such as GCGCGC, and indeed the G and C nucleotides are in different conformations,
leading to the zig-zag pattern. The big difference is at the G nucleotide. It has the sugar in the C3'
endoconformation (like A-form nucleic acid, and in contrast to B-form DNA) and the guanine
base is in the synconformation. This places the guanine back over the sugar ring, in contrast to the
usual anticonformation seen in A- and B-form nucleic acid. Note that having the base in the
Z-form DNA
anticonformation places it in the position where it can readily form H-bonds with the
complementary base on the opposite strand. The duplex in Z-DNA has to accomodate the
distortion of this G nucleotide in the synconformation. The cytosine in the adjacent nucleotide of
Z-DNA is in the "normal" C2' endo, anticonformation.Even classic B-DNA is not completely
uniform in its structure. X-ray diffraction analysis of crystals of duplex oligonucleotides shows that
a given sequence will adopt a distinctive structure. These variations in B-DNA may differ in the
propeller twist (between bases within a pair) to optimize base stacking, or in the 3 ways that 2
successive base pairs can move relative to each other: twist, roll, or slide.
Refers to the orientations of the two single strands that
compose a double-stranded DNA helix. Strands are
Anti-parallel
oriented such that one strand's 5' end is directly across
from the other strand's 3' end.
In DNA, refers to the nitrogen-carbon linkage between the 9' nitrogen of purine bases or 1'
Conformation at the glycosidic bond
nitrogen of pyrimidine bases and the 1' carbon of the sugar group.
a groove that spirals around the DNA double helix; provides a location where a protein can bind
to a particular sequence of bases and affect the expression of a gene
Major groove In a helix, refers to the larger of the unequal grooves that are formed as a result of the double-
helical structure of DNA. As a result of the patterns of hydrogen bonding between
complementary bases of DNA, the sugar groups stick out at 120 degree angles from each other
instead of 180. The major groove is generated by the larger angular distance between sugars.
In a helix, refers to the smaller of the unequal grooves that are formed as a result of the double-
helical structure of DNA. As a result of the patterns of hydrogen bonding between
Minor groove
complementary bases of DNA, the sugar groups stick out at 120 degree angles from each other
instead of 180. The minor groove is generated by the smaller angular distance between sugars.
Purine
One of two categories of nitrogen base ring compounds found in DNA and RNA. A purine is a
nine-membered double ring composed of one five-membered joined to a six membered ring
Purines
containing four nitrogens
Cytosine and Thymine
Pyrimidines
One of two categories of nitrogen base ring compounds found in DNA and RNA. A six-
membered ring containing two nitrogens
anti-pyrimidine (T)
anti-purine (A)
conformation at glycosidic bond syn-pyrimidine (T)
syn-purine (A)
All the nucleotides in A and B-DNA are anti-

To understand the structure of B-form DNA and numerous structural variations in the DNA helix, it
is important to have an appreciation of the individual components of DNA. DNA is composed of
aromatic bases (a purine or pyrimidine ring), ribose sugars, and phosphate groups. The many
variations in the structures of the bases and the sugars, and in the structural relationship of the
base to the sugar, give rise to differences in the helical structure of DNA
Purines: Adenine and Guanine

Two different heterocyclic aromatic bases with a purine ring (composed of carbon and nitrogen)
are found in DNA. The numbering system for the purine ring is shown in Figure 1.2. The two
common purine bases found in DNA, adenine and guanine, are also shown in Figure 1.2. These are
synthesized in cells de novo in multistep biochemical reactions with the base being built on a
phosphorylated ribose sugar molecule. The last common interme- diate in their synthesis is
inosine. Adenine has an amino group (-NH2 ) on the C6 position of the purine ring (carbon at
position 6 of the purine ring). Guanine has an amino group at the C2 position and a carbonyl
Structures of purine and pyrimidine bases.
group at the C6 position.
Pyrimidines: Thymine, Cytosine, and Uracil

The two pyrimidine bases commonly found in DNA are thymine and cytosine. These are also
synthesized in cells de novo in multistep reactions. The structures of the basic six-member ring,
thymine, and cytosine are shown in Figure 1.2. Thymine contains a methyl group at the C5 position
with carbonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at the C5
position and an amino group at C4.
Uracil is similar to thymine but lacks the methyl group at the C5 position (Figure 1.2). Uracil is not
usually found in DNA. It is a component of ribonucleic acid (RNA) in which it is utilized in place
of thymine as one of the pyrimidines. RNA also differs from DNA in the structure of the sugar
moiety, as described later.
The purines and pyrimidines are well suited to their roles as the informational molecules of the
cell. The differential placement of hydrogen bond donor and acceptor groups gives the bases
the unique structural identity that allows them to serve as the genetic information. The hydrogen
Purines and Pyrimidines as Informational atoms of amino groups provide hydrogen bond donors, whereas the carbonyl oxygens and ring
Molecules nitrogens provide hydrogen bond acceptors. The aromatic nature of the rings means that they are
rigid planar molecules. This flatness is important in the organization of bases within the helix,
since it allows the bases to stack uniformly within the helix. As described subsequently, this
stacking helps protect the chemical identity of the bases

In DNA a slightly different sugar, f3-o-2-deoxyribose, is found. This is a derivative of f3-o-ribose
in which the hydroxyl (-OH) at the 2' position is replaced by a hydrogen (-H). Biochemically this
is done by the enzyme ribonu- cleotide reductase which converts all ribonucleoside
diphosphates (or occa- sionally triphosphates) in a chemical reduction reaction from 2' OH to 2'
H.
The sugar moiety of DNA is one of the more flexible and dynamic parts of the molecule. Figure
1.4 shows the structures of the common sugar conformations that are found in the various forms
of DNA. The sugar ring structure is easy to envision if one thinks of an envelope. In the envelope
form, the four carbons form a plane at the corners of the body of the envelope. The oxygen is at
Deoxyribose Sugar Is Found in DNA
the position representing the top of the envelope flap. The oxygen can be bent out of the plane
of the body of the envelope. Twisting the C2' and C3' carbons relative to the other atoms results
in various twist forms of the sugar ring. To form the C2' endo form of the ribose sugar, C2' twists
up from the plane of the four carbons. To form the C3' endo, C3' twists down out of the plane of
the four carbons.
Dideoxyribonucleotides are used in DNA sequencing reactions. A 2',3' dideoxyribonucleotide has
hydrogen atoms at both the 2' and 3' positions (see Figure 1.5). When incorporated into a DNA
chain, the dideoxyribonucleotide blocks further polymerization, since there is no 3' OH to which
another base can be added. This will become apparent from the ensuing discussion of the
phosphodiester bond and polynucleotides

watson and Crick first described the structure of the DNA double helix in 1953. A representation
of their model is shown in Figure 1.1. Duplex DNA is a right-handed helix formed by two individual
DNA strands aligned in an antiparallel fashion. This means that one strand is oriented in the 5'
~3'direction and the other in the 3'~5' direction. The two strands are held together by hydrogen
bonds between individual bases. The bases are stackednear the center of the cylindrical helix.
The base stacking provides consider- able stability to the double helix. The sugar and phosphate
groups are on the outside of the helix and form a "backbone" for the helix. There are about 10
base pairs (bp) per turn of the double helix.
Two important pieces of information were critical for the development of this structure, one of
which was "Chargaff's Rules." In the early 1950s, Chargaff pointed out that the amount of adenine
always equalled the amount of thymine and the amount of guanine always equalled the amount
of cytosine (Chargaff, 1951; Zamenhof et al., 1952). This was true for DNA puri- fied from a wide
variety of organisms, and true regardless of the total G + C (or A + T) content. This condition is met
The Structure of Double-Stranded DNA by having two strands of DNA in which the bases are hydrogen bonded with strict
complementary base pairing. Specifically, A only pairs with T (A·T) and G only pairs with C (G· C).
(See Table 1.1 for conventions in designating polynucleotide chains and base pairs.) The second
piece of information came from X-ray diffraction patterns of DNA fibers (Wilkins and Randall,
1953; Wilkins et al., 1953) which showed that the geometric shape of DNA is a right-handed helix.
Watson and Crick used this information to deduce a model for the structure of DNA.
The double-helical model of DNA seems simple and straightforward. However, the elucidation of
this structure was not trivial. Watson and Crick had no way of knowing that DNA was necessarily
composed of two strands, that these strands were antiparallel, or that the bases were paired
exclusively A . T and G· C. In addition, because of tautomerization and ionization, Watson and
Crick were not even sure of the chemical form of the bases. This is a very important point because
tautomerization and ionization change the electronic configuration of the bases, which changes
their base-pairing properties. Moreover, as discussed subsequently, there are many different
ways to form hydrogen bonds between two bases

Watson-Crick model DNA. Deep, wide major groove.
The structure of B-form DNA, the most common form, was originally deduced from X-ray
diffraction analysis of the sodium salt of DNA fibers at 92% relative humidity (Langridge et al.,
1960a,b). B-Form DNA is pictured in Figure 1.12, where various helix parameters and features are
indicated. A molecular model is shown in Figure 1.14. There are about 10.5 bp per right- handed
helical turn in B-DNA (helix parameters are listed in Table 1.3). The form of the ribose sugar is C2'
endo. The term B-form DNA will be used to refer to the right-handed helical form commonly
found for DNA in solution.
A dominant feature of B-form DNA is the presence of two distinct grooves, a major and a minor
groove, shown in Figures 1.12 and 1.15. These two grooves obviously provide very distinct surfaces
B- FORM DNA
with which proteins can interact. As discussed in Chapter 8, different DNA binding proteins have
domains that interact with either the major or the minor groove. Certain chemi- cals and drugs
can interact specifically with either the major or the minor groove. Different functional groups on
the purine and pyrimidine bases are accessible from the major or the minor groove (Figure 1.15).
The Watson-Crick hydrogen bonding surfaces are not available to solvent or proteins, since the
functional groups involved in hydrogen bonding are interacting with each other (in
complementary base pairs) at the center of the double helix. The Hoogsteen hydrogen bonding
surface of purines is accessible through the major groove. This is evident by looking down the
axis of the double helix as shown in the representation in Figure 1.14. In this projection, the stacked
base pairs form a central core surrounded by the phosphate backbone. The center of the helix
is a relatively chemically inert place to store genetic information.
Condensed form of DNA. Deeper major groove and shallower minor groove.
A-Form DNA was originally identified by X-ray diffraction analysis of DNA fibers at 75% relative
humidity (Fuller et al., 1965). The structure of A- DNA is shown in Figure 1.16 and the helix
parameters are listed in Table 1.3. The grooves are not as deep as in B-DNA, and the bases are
much more tilted (to 20°). Another significant difference between A-form DNA and B-form DNA is
that the sugar pucker is C3' endo (compared with C2' endo for B-DNA).
A- form DNA
Does A-DNA exist in biological systems? Runs of homopurine· ho- mopyrimidine DNA sequence
[poly(dG) . poly(dC), for example] seem to set up an A-like helix, as determined by characteristic
circular dichroism (CD) spectra (Fairall et al., 1989). Therefore, it is reasonable to assume that
within a generally B-like DNA molecule, specific regions may exist in an A-DNA form. This would
be a function of sequence composition of DNA (see Section E). RNA frequently exists in a
double-helical form in transfer RNAs (tRNA), ribosomal RNAs (rRNA), and parts of messenger
RNAs (mRNA). Double- stranded RNA forms an A-like helix. The ribose configuration for double-
stranded RNA is C3' endo, which is a distinguishing feature of the A-DNA helix.

The plane of the base is almost perpendicular to that of the sugars and approximately bisects the
O4’-C1’-C2’ angle. This allows the bases to occupy either of two principal orientations. The anti
conformer has the smaller H-6 (pirimidine) or H-8 (purine) atom above the sugar ring, while the
syn conformer has the larger O-2 (pirimidine) or N-3 (purine) in that position. Pirimidines occupy a
narrow range of anti conformations, while purines are found in wider range of anti conformations.
There is only one known case where a purine adopts a syn conformation. The unusual form is to
find in an even more unusual structure namely left handed Z-DNA. An example of possible
interaction between guanosines in syn and anti conformation is shown on a drawing below
Two other bonds which are important for determining the DNA structure are:
syn and anti conformation the bond between C4’ and C5’ which defines the position of the 5’phosphate relative to the sugar
ring (the favoured conformations of this bond are synclinical and antiperiplanar)
C-O and P-O bonds (the former usually appears in antiperiplanar conformation and the latter in
gauche conformation.
Syn addition occurs when H2 reacts with a double bond. In this type of a reaction, both hydrogen
atoms are added to the same side. The product that puts the hydrogen atoms on opposite sides
doesn't form, like you can see here:The reason has to do with the mechanism for adding
hydrogen to an alkene. We're actually first plating hydrogens onto a thin metal sheet (the Pd-C).
This plating keeps the hydrogen atoms from freely rotating around the molecule, so they must be
added to the same side of the molecule
syn and anti addition are different ways in which two substituents can be added to a double
bond or triple bond.
Syn addition is the addition of two substituentsto the same side (or face) of a double bond or
syn ( same ) and anti ( opposite) triple bond, resulting in a decrease in bond order but an increase in number of substituents.
In anti addition, two substituents are added to opposite sides (or faces) of a double bond or
triple bond, once again resulting in a decrease in bond order and increase in number of
substituents

In a DNA double helix, each type of nucleobase on one strand bonds with just one type of
nucleobase on the other strand. This is called complementary base pairing. Purines form
hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds,
and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two
nucleotides binding together across the double helix is called a Watson-Crick base pair. DNA
with high GC-content is more stable than DNA with low GC-content. A Hoogsteen base pair is a
rare variation of base-pairing.[27] As hydrogen bonds are not covalent, they can be broken and
rejoined relatively easily. The two strands of DNA in a double helix can thus be pulled apart like a
zipper, either by a mechanical force or high temperature.[28] As a result of this base pair
complementarity, all the information in the double-stranded sequence of a DNA helix is
duplicated on each strand, which is vital in DNA replication. This reversible and specific
interaction between complementary base pairs is critical for all the functions of DNA in
organisms.
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two
nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA
double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific
hydrogen bonding patterns, "Watson–Crick" base pairs (guanine–cytosine and adenine–thymine)
[1] allow the DNA helix to maintain a regular helical structure that is subtly dependent on its
nucleotide sequence.[2] The complementary nature of this based-paired structure provides a
redundant copy of the genetic informationencoded within each strand of DNA. The regular
structure and data redundancy provided by the DNA double helix make DNA well suited to the
storage of genetic information, while base-pairing between DNA and incoming nucleotides
provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase
transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing
patterns that identify particular regulatory regions of genes.
Watson-Crick base pairing
Intramolecular base pairs can occur within single-stranded nucleic acids. This is particularly
important in RNA molecules (e.g., transfer RNA), where Watson–Crick base pairs (guanine–
cytosine and adenine–uracil) permit the formation of short double-stranded helices, and a wide
variety of non-Watson–Crick interactions (e.g., G–U or A–A) allow RNAs to fold into a vast range
of specific three-dimensional structures. In addition, base-pairing between transfer RNA (tRNA)
and messenger RNA(mRNA) forms the basis for the molecular recognition events that result in
the nucleotide sequence of mRNA becoming translated into the amino acid sequence of proteins
via the genetic code.
The size of an individual gene or an organism's entire genome is often measured in base pairs
because DNA is usually double-stranded. Hence, the number of total base pairs is equal to the
number of nucleotides in one of the strands (with the exception of non-coding single-stranded
regions of telomeres). The haploid human genome (23 chromosomes) is estimated to be about
3.2 billion bases long and to contain 20,000–25,000 distinct protein-coding genes.[3][4][5] A
kilobase (kb) is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or
RNA.[6] The total number of DNAbase pairs on Earth is estimated at 5.0×1037 with a weight of 50
billion tonnes.[7] In comparison, the total mass of the biosphere has been estimated to be as
much as 4 TtC (trillion tons of carbon)
(There are other kinds of basepairs, with H-bonds between different atoms in the
purines/pyrimidines. These are only found in unusual DNA structures; for example, Hoogsteen
basepairs, found in quadruplex DNA etc. But we won’t be saying much about them in this course.)
The two Watson-Crick basepairs have very similar sizes

and shapes, allowing them to fit in any sequence into a
quite uniform double helix structure
"major groove" and "minor groove" indicate where the

edges of the basepairs end up in the double helix.
base pairs
A-T = space floor representation
hydrogen atoms don't show up
B- Nitrogen
R- oxygen

because they're flat they are hydrophobic and to escape
from water they like to stack together ( minimise exposure
of surface of bp to water )
The flat surfaces of the basepairs (i.e. their 'top'and

'bottom’) are hydrophobic ('water-hating'). To escape from
the water they like to stack on top of each other.
they'll come as close as together until van Der waals

repels them
Base stacking is a common arrangement of nucleobases

found in the three dimensional structure of nucleic acids.
Basepair stacking Bases (or base pairs) are planar, and these planes stack at
contact distance (about 3.4 Angstrom), excluding water
and maximizing Van der Waals interactions. In terms of
structural stability of nucleic acids in aqueous solution, the
stacking interactions of bases play a larger role than the
hydrogen bonds formed by the bases
DNA double helix: In double-stranded DNA, bases from

two strands pair up to form base pairs, which are stacked
along the helix axis of the double strand. Zooming in to a
detailed view of a G:C base pair, the extent of the
stacking contacts are determined by the sequence. G:C
base pairs contribute more to the thermal stability of DNA
than A:T base pairs because they stack better.

These interactions are largely ionic interactions, with partial charges on nearby atoms attracting
one another. Hydrogen bonds are responsible for specific base-pair formation in the DNA
double helix. The hydrogen atom in a hydrogen bond is partially shared by two electronega- tive
atoms such as nitrogen or oxygen. The hydrogen-bond donor is the group that includes both the
atom to which the hydrogen atom is more tightly linked and the hydrogen atom itself, whereas
the hydrogen-bond acceptor is the atom less tightly linked to the hydrogen atom (Figure 1.9). The
electro- negative atom to which the hydrogen atom is covalently bonded pulls electron density
away from the hydrogen atom, which thus develops a partial positive charge (d􏲐). Thus, the
hydrogen atom with a partial positive charge can interact with an atom having a partial negative
hydrogen bonds charge (d􏲏) through an ionic interaction.
Hydrogen bonds are much weaker than covalent bonds. They have energies ranging from 4 to
20 kJ mol􏲏1 (from 1 to 5 kcal mol􏲏1). Hydrogen bonds are also somewhat longer than covalent
bonds; their bond lengths (measured from the hydrogen atom) range from 1.5 Å to 2.6 Å; hence, a
distance ranging from 2.4 Å to 3.5 Å separates the two nonhydrogen atoms in a hydrogen bond.
The strongest hydrogen bonds have a tendency to be approximately straight, such that the
hydrogen-bond donor, the hydrogen atom, and the hydrogen-bond acceptor lie along a straight
line. This tendency toward lin- earity can be important for orienting interacting molecules with
respect to one another. Hydrogen-bonding interactions are responsible for many of the
properties of water that make it such a special solvent, as will be described shortly.
ThebasisofavanderWaalsinteractionis that the distribution of electronic charge around an atom

fluctuates with time. At any instant, the charge distribution is not perfectly symmetric. This
transient asymmetry in the electronic charge about an atom acts through ionic interactions to
induce a complementary asymmetry in the electron distribution within its neighboring atoms. The
atom and its neighbors then attract one another. This attraction increases as two atoms come
closer to each other, until they are separated by the van der Waals contact distance (Figure 1.10).
At distances shorter than the van der Waals contact distance, very strong repulsive forces
become dominant because the outer electron clouds of the two atoms overlap.
van deer Waals
Energies associated with van der Waals interactions are quite small; typical interactions contribute
from 2 to 4 kJ mol􏲏1 (from 0.5 to 1 kcal mol􏲏1) per atom pair. When the surfaces of two large
molecules come together, however, a large number of atoms are in van der Waals contact, and
the net effect, summed over many atom pairs, can be substantial.

Water is the solvent in which most biochemical reactions take place, and its properties are
essential to the formation of macro- molecular structures and the progress of chemical reactions.
Two properties of water are especially relevant:
1. Water is a polar molecule. The water molecule is bent, not linear, and so the distribution of
charge is asymmetric. The oxygen nucleus draws elec- trons away from the two hydrogen nuclei,
which leaves the region around each hydrogen atom with a net positive charge. The water
molecule is thus an electrically polar structure.
Properties of water 2. Water is highly cohesive. Water molecules interact strongly with one another through hydrogen
bonds. These interactions are apparent in the structure of ice (Figure 1.11). Networks of hydrogen
bonds hold the structure together; similar interactions link molecules in
liquid water and account for many of the properties of water. In the liquid state, approximately
one in four of the hydrogen bonds present in ice are broken. The polar nature of water is
responsible for its high dielectric constant of 80. Molecules in aqueous solution interact with
water molecules through the formation of hydrogen bonds and through ionic interactions. These
interactions make water a versatile solvent, able to readily dissolve many species, especially polar
and charged compounds that can participate in these interactions.
A final fundamental interactioncalled the hydrophobic effect is a manifestation of the proper-ties

of water. Some molecules (termed nonpolar molecules)cannot participate in hydrogen bonding
or ionic interac-tions. The interactions of nonpolar molecules with watermolecules are not as
favorable as are interactions betweenthe water molecules themselves. The water molecules
incontact with these nonpolar molecules form “cages” aroundthem, becoming more well ordered
than water molecules free in solution. However, when two such nonpolar molecules come
together, some of the water molecules are released, allowing them to interact freely with bulk
the hydrophobic effect
water (Figure 1.12). The release of water from such cages is favorable for reasons to be considered
shortly. The result is that nonpolar molecules show an increased tendency to associate with one
another in water compared with other, less polar and less self-associating, solvents. This tendency
is called the hydrophobic effect and the associated interactions are called hydrophobic
interactions.

First, each phosphate group in a DNA strand carries a negative charge. These negatively charged
groups interact unfavorably with one another over distances. Thus, unfavorable ionic
interactions take place when two strands of DNA come together. These phosphate groups are far
apart in the double helix with distances greater than 10 Å, but many such interactions take place
(Figure 1.13). Thus, ionic interactions oppose the formation of the double helix. The strength of
these repulsive ionic interactions is dimin- ished by the high dielectric constant of water and the
presence of ionic species such as Na or Mg2 ions in solution. These positively charged species
interact with the phosphate groups and partly neutralize their negative charges.
Second, as already noted, hydrogen bonds are important in determining the formation of specific
base pairs in the double helix. However, in single- stranded DNA, the hydrogen-bond donors and
acceptors are exposed to solution and can form hydrogen bonds with water molecules. When
two single strands come together, these hydrogen bonds with water are broken and new
hydrogen bonds between the bases are formed. Because the number of hydrogen bonds broken
is the same as the number formed, these hydrogen bonds do not contribute substantially to
driving the overall process of double-helix formation. However, they contribute greatly to the
specificity of binding. Suppose two bases that cannot form Watson-Crick base pairs are brought
together. Hydrogen bonds with water must be broken as the bases come into contact. Because
The double helix is an expression of the rules of the bases are not complementary in structure, not all of these bonds can be simultaneously
chemistry replaced by hydrogen bonds between the bases. Thus, the formation of a double helix between
noncomplementary sequences is disfavored.
Third, within a double helix, the base pairs are parallel and stacked nearly on top of one another.
The typical separation between the planes of adjacent base pairs is 3.4 Å, and the distances
between the most closely approaching atoms are approximately 3.6 Å. This separation distance
cor- responds nicely to the van der Waals contact distance (Figure 1.14). Bases tend to stack even
in single-stranded DNA molecules. However, the base stacking and associated van der Waals
interactions are nearly optimal in a double-helical structure.
Fourth, the hydrophobic effect also contributes to the favorability of base stacking. More-
complete base stacking moves the nonpolar surfaces of the bases out of water into contact with
each other.
The principles of double-helix formation between two strands of DNA apply to many other
biochemical processes. Many weak interactions contribute to the overall energetics of the
process, some favorably and some unfavorably. Furthermore, surface complementarity is a key
feature: when complementary surfaces meet, hydrogen-bond donors align with hydrogen- bond
acceptors and nonpolar surfaces come together to maximize van der Waals interactions and
minimize nonpolar surface area exposed to the aqueous environment. The properties of water
play a major role in determining the importance of these interactions
What holds the DNA in a double helix structure?
Building the double helix

basepairing of complementary strands basepair stacking phosphate charge repulsion preferred

conformations of the deoxyribose and phosphate bonds
force driving apart two ions, molecules, or regions of
molecules of the same electric charge
The DNA “backbone” has one negative charge on each

phosphate group – so DNA is highly negatively charged
overall in typical biological conditions!
(at very low pH, the phosphates are protonated; that’s
Charge repulsion
whyit’s called deoxyribonucleic acid. At typical cell pH,
the negative charges are balanced by positive ions such
as Na+ or Mg2+, diffusely in solution around the DNA
molecule.)
The negative charges repel each other, and the charged
phosphates are hydrophilic; hence they’ll prefer to be on
the outside of the DNA structure
Deoxyribose 'sugars' are in C-2'-endoconformation

Bases are in anti-conformation All basepairs are Watson-
Crick
Helix pitch is the length of one complete helical turn of

DNA. In textbook Bform DNA, one helical turn of 10 bp is
completed in 34 A.
Base pair tilt refers to the angle of the planar bases with
respect to the helical axis. The tilt angle is measured by
considering the angle made by a line drawn through the
two hydrogen bonded bases relative to a line drawn
Dimensions of 'ideal' B-DNA
perpendicular to the helix axis. This is illustrated in Figures
1.12, 1.13, and 1.14. A base pair that was perfectly flat, that is,
perpendicular to the helix axis, would have a tilt angle of
0°. In Bcform DNA the bases are tilted by only- 6°.In
A·form DNA the base pairs are significantly tilted at an
angle of 20°

Early structural studies identified two slightly different
forms of DNA double helix, A and B.DNA in cells is mostly
much like B-DNA (as on the previous slides). A-DNA is a
bit 'shorter and fatter', and major and minor grooves are
more equal in width than in B-DNA. Some sequences of
B-DNA and A-DNA natural DNA might look like this, or somewhere between B
and A.
[For most purposes it's simplest to regard B-DNA as the

'standard' DNA structure. On Day 2 we'll discuss how real
DNA deviates from ideal B-DNA.]
Real DNA is almost never exactly like the 'ideal' B structure. Its character isaffected by its basepair
sequence, and by external factors like ions and supercoiling.
Examples:
The width of the minor groove varies (e.g. GC-rich wide, AT-rich narrow) Helix twist per basepair
varies quite a lot
A brief summary on structural variation Basepairs can be distorted, and don’t always sit flat on each other The DNA axis can bend
etc.
These variations have important implications for genome structure and stability, and for protein-
DNA interactions. More about these issues on Day 2!
AND... Cellular DNA is usually covered with DNA-binding proteins! Binding of proteins can
change DNA structure dramatically. More on Days 3 and 4

These 'unusual' forms of DNA may be important in some biological events. We'll mention some of
them later in the course.
At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The
main function of these regions is to allow the cell to replicate chromosome ends using the
enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′
ends of chromosomes.[58] These specialized chromosome caps also help protect the DNA ends,
and stop the DNA repair systems in the cell from treating them as damage to be corrected.[59] In
human cells, telomeres are usually lengths of single-stranded DNA containing several thousand
repeats of a simple TTAGGG sequence.
These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked
sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four
guanine bases, known as a guanine tetrad, form a flat plate. These flat four-base units then stack
on top of each other to form a stable G-quadruplex structure.[62] These structures are stabilized
A 'zoo' of unusual DNA structures by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre
of each four-base unit.[63] Other structures can also be formed, with the central set of four bases
coming from either a single strand folded around the bases, or several different parallel strands,
each contributing one base to the central structure.
In addition to these stacked structures, telomeres also form large loop structures called telomere
loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by
telomere-binding proteins.[64] At the very end of the T-loop, the single-stranded telomere DNA
is held onto a region of double-stranded DNA by the telomere strand disrupting the double-
helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a
displacement loop or D-loop.
Z-DNA is a left-handed helix that is very different from right-handed DNA forms. Z-DNA can form
in alternating purine-pyrimidine tracts under certain conditions, including high salt, the presence
of certain divalent cations, or DNA supercoiling. Compared with B-DNA, there are major struc-
tural differences in the sugar pucker, rotations about the glycosidic bond, and orientation of base
pairs within the helix (Table 1.3)

The two strands of DNA are held together by many weak
interactions (mainly H-bonds). The strands can come apart
(melt) quite easily. Proteins can do this (e.g. helicases, RNA
polymerase etc.), but it can also happen at high
temperature, in unusual chemical conditions or by using
chemical denaturants such as formamide.
Denaturation ("melting") of DNA
"Melting temperature" of DNA depends on the AT/GC
content, and salt concentration, pH, etc.
The reverse of melting is called annealing

The double-helical structure of DNA is remarkably stable.
This stability is derived from two chemical forces,
hydrogen bonding and base stacking interactions, as
discussed in Section C,1. In addition, the helix is solvated
or covered with water molecules which form a "shell of
hydration" around the DNA. To melt the two strands or
denature the DNA, all these stabilizing forces must be
overcome.
The two strands of DNA come apart readily on incubation
at pH > 12 or pH < 2 due to ionization of the bases (Figure
1.19). As discussed earlier, ionization results in a change in
the hydrogen bond donor/acceptor properties of the
bases, which will disrupt the normal A . T and C . G
Watson-Crick hydrogen bonds. In addition, the shell of
hydration surrounding the DNA is disrupted at very high
or low pH, destabilizing base stacking. Acid treatment of
DNA leads to depyrimidation and depurination, the loss of
bases by cleavage of the glycosidic bond. The
phosphodiester bond is more susceptible to hydrolysis
at these abasic sites. Thus, strong acid will degrade DNA.
Since single strands of DNA are relatively stable in alkali,
denaturation is usually ac- complished by alkali treatment.
Increasing the temperature of DNA destabilizes the

double helix, resulting in the separation of the two
strands. Heat both disrupts the hydrogen bonds and
destroys the shell of hydration of DNA leading to a loss of
forces holding the two strands together. Experimentally,
there is a linear relationship between the G + C content
and the melting temperature of DNA (Marmur and Doty,
1962). The temperature at which 50% of a DNA sample is
melted is called the melting temperature or Tm- Moreover,
there is a linear relationship between the Tm of DNA and
the calculated stacking energies (listed in Table 1.2). This
2. Denaturation and Renaturation suggests that the thermal stability of DNA is a function of
base stacking, not only hydrogen bonding (see Saenger,
1984, for more discussion).
DNA denaturation can be measured or monitored in many
different ways. One method involves measurement of a
characteristic increase in the absorbance at 260 nm called
hyperchromicity, which results from the un- stacking of the
bases. Another method employs enzymes specific for
single- stranded DNA (such as Sl nuclease). Selective
binding reactivity of single- or double-stranded DNAs
with various surfaces can also be used. For example,
hydroxylapatite, a C a P 0 4 precipitate, will bind double-
stranded DNA but not single-stranded DNA at certain
phosphate concentrations.
Rapid removal of denaturing conditions, such as quickly
cooling a heat- denatured DNA sample, results in the
collapse of single strands into an un-ordered random coil
(Figure 1.19). In this configuration the two strands cannot
reform a double helix. However, if DNA is slowly cooled,
interstrand nucleation can occur in which small
complementary regions on the two opposite DNA
strands come together and form a short double-helical
region. Once nucleation has occurred, the rest of the
double helix renatures very rapidly. The rate limiting step
in renaturation is the nucleation event.
Negative DNA supercoiling, which is discussed in Chapter
3, can also drive the melting of a region of DNA.
Supercoiled DNA contains a great deal of free energy that
can be used to transiently or stably melt local regions of
supercoiled DNA. Not surprisingly, these are usually
regions of DNA rich in A + T.
Denaturation and renaturation of DNA. The double helical configuration of DNA can be
denatured by heat, alkali, or acid. In this process the two strands separate as base stacking and
hydrogen bonding interactions are disrupted. Ifone quickly removes the heat or neutralizes the
DNA solution (quick cool or neutralize), the DNA will collapse into a compact random coil in
denaturation which some bases are hydrogen bonded. If a denatured solution of DNA is slowly cooled, the
two single strands can reform a paired double helical molecule. The process requires a
nucleation event in which a region of complementary bases on opposite strands finds each other,
comes into register, and begins to form a hydrogen bonded double helix with stacked base pairs.
Once nucleation has occurred, the rest of the DNA molecule rapidly renatures.
DNA REPLICATION

DNA polymerases are responsible for replicating the DNA. They syn- thesize a new strand of
DNA using a preexisting strand as a template. They utilize nucleoside triphosphates adding a 5'
mononucleotide to the 3' OH end of the nascent DNA chain. Thus, polymerization occurs in the 5'
to 3' direction. Because of the 5' to 3' directionality of the polymerase, one strand of a
replication fork, the leading strand, can be replicated continuously. The other strand, the lagging
strand, must be replicated in short "Okazaki" pieces. There are three polymerases in Escherichia
coli called pol I, pol II, and pol III which are encoded by the polA, dinA, and pol C (or dnaE)
genes, respec- tively. In E. coli, pol III is primarily responsible for replication of the chromo-
some. pol I is involved in joining the short Okazaki pieces. pol I and pol II are involved in DNA
repair functions. In eukaryotic cells there are three nuclear polymerases (a, 13, and 8) and one
mitochondrial polymerase 'Y. Polymerase a is believed to replicate the leading strand, 8 may
replicate the lagging strand, and 13 is involved in DNA repair. These enzymes and the process of
DNA replication are described in more detail by Kornberg and Baker (1992).
Primase is a required enzyme because DNA polymerase does not initiate synthesis de novo on a
DNA replication
single-stranded DNA template. DNA polymerase must have a primer on which to add 5'
mononucleotides. The primer, which must be base-paired to the template, can be a short piece of
either DNA or RNA with a 3' OH end. RNA polymerase can begin synthetis de novo of RNA
complementary to a strand of DNA. A specialized RNA polymerase called primase, encoded by
dnaG in E. coli, makes the primer for synthesis by DNA polymerase III.
DNA helicases are proteins that move down the DNA separating the two strands or denaturing
the double helix. Helicases require energy from ATP for their activity. Helicases work ahead of
DNA polymerase, separating the strands for polymerase. There are helicases with two different
polarities, those track along a strand in the 3' to 5' direction and those that track from 5' to 3'.
DNA ligase is an enzyme that forms a covalent phosphodiester bond between a 3' OH and 5' P04
at the ends of two polynucleotide chains (frequently at a nick in one strand of a double helix).
Ligase requires energy from ATP to seal the DNA. DNA ligase is used extensively in DNA cloning
methodologies
Two crucial features of DNA polymerases:(1) DNA

polymerase requires a primer. It will only add nucleotides
onto the end of an existing strand; it can’t start a new one.
(2) DNA polymerase requires a template strand. The
nucleotide to be added must be complementary (i.e.
make a Watson-Crick basepair) to the next base in the
DNA REP
template.
reactions that DNA polymerase catalyses

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA
molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are
essential for DNA replication and usually work in groups to create two identical DNA duplexes
from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA
strands to create two new strands that match the existing ones.[1][2][3][4][5][6] These enzymes
catalyze the chemical reaction
deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1.
reaction DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at
a time. Every time a cell divides, DNA polymerases are required to duplicate the cell's DNA, so
that a copy of the original DNA molecule can be passed to each daughter cell. In this way,
genetic information is passed down from generation to generation.
Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its
tightly woven form, in the process breaking the hydrogen bonds between the nucleotide bases.
This opens up or "unzips" the double-stranded DNA to give two single strands of DNA that can
be used as templates for replication in the above reaction.
In E. coli, the main DNA polymerase is PolIII, a large

complex with 18 protein subunits. Another important E.
coli DNA polymerase, PolI, is mainly used for DNA repair.
The PolIII 'replisome' replicates the circular E. coli
chromosome. It starts at the 'origin of replication' oriC, a
specific place in the chromosome DNA.
A sum: E. coli chromosome is 4.7 x 106 bp. It's replicated in
40 minutes, i.e. 2400 sec. Replication from oriC is
bidirectional, i.e. one PolIII complex sets of in each
direction along the DNA. So, the rate of PolIII = 4.7 x 106/2
x 2400 bp replicated per second,= ~1000 bp (or 2000
bacterial dna polymerase
nt)/second!
Eukaryote replication forks are reported to go slower –

~1500 nt/minute.
This multi-protein + DNA complex is called the ‘replisome
ter opposite oric / origin is bidirectional and meet at terms

region and finish replication

The 'core' eukaryote replisome contains more than 30 different proteins, but many more proteins
join in as replication proceeds.
There are at least 3 polymerase enzymes at the replication fork (each enzyme is a tetramer of
four different subunits).
Polα (includes primase, and extends RNA primers ~10-12 nt); Polε (makes leading strand)Polδ
(makes lagging strand)
Eukaryotes The specific functions of each Pol in replication are still quite controversial
In total, humans have about 20 different DNA polymerases!
Others
Many phages, viruses and transposons encode their own DNA polymerases.
And we mustn't forget reverse transcriptase - a DNA polymerase that uses RNA as a template.
more extra proteins that join while various things are happening / time wise //
The functions of the proteins in the replisome are

conserved through all living organisms (though the
Conservation of replication proteins proteins may be unrelated). For example:
see table imp
"Cartoon" representation of a DNA polymerase - shows

the new nucleotide to be added, sitting at the active site
Most polymerases have a 'proofreading' activity; a3'-5'

exonuclease which removes the most recently added
DNA polymerase catalytic subunit structures
nucleotide if it does not base-pair correctly with the
template
-shows how 3' end can flip into the Exo active site when a
wrong nucleotide is added.

E. coli PolIII is highly processive (i.e., puts on many
thousands of nucleotides without falling off the DNA).
Processivity requires the ‘sliding clamp’ β-subunit (as a
dimer). stick to each other at two surfaces that makes a
hole that dna will pass and DNA polymerase around // tie
polymerase on DNA so it docent escape //
β2 has a doughnut/polomint-like structure. It seems to be
designed not to make any specific contacts with the DNA
'Sliding clamps' of DNA polymerases
as it slides along it.
In mammalian polymerases, the sliding clamp is PCNA
('proliferating cell nuclear antigen') which is a trimer, but its
structure is very similar to the β2 dimer!
Each β subunit has 3 almost identical domains, and each

domain is roughly 2-fold symmetric. The hole is lined with
12 α-helices; DNA fits easily into it.
To load the sliding clamp (β2) onto DNA, the ring has to
be split open. This is done by the γ-complex, and requires
ATP hydrolysis. Details are described in Pomerantz et al.
2007.
Structure of β2 dimer + DNA:
dna in the middle of the dimer ring
alpha helices - smooth surfaces
how? // rung has to be split the dimers to let dna inside it

and that's done by gamma complex and it requires atp
hydrolysis
Each PolIII holoenzyme comprises two polymerase

functional units; one makes the leading strand
continuously, the other makes the lagging strand
discontinuously in 1 - 2 kbp Okazaki fragments.
Replication of leading and lagging strands (E. coli)
replisome contains 2 polymerase functional units - leading

and lagging strand holds on the black beta dimer // the
clamp // polymerase adds nucleotides //
It's probably most realistic to think of the proteins as being
fixed in one place in the cell, so that the DNA is being
at the replication fork
passed through a stationary replication complex
check saved website // all good notes
What do we mean by the following terms: template; primer; leading strand; lagging strand;
semiconservative replication; replisome; polymerase; helicase; proof-reading?
Why does DNA replication need two polymerases at the replication fork, that do different things?
revision What are Okazaki fragments, how do they get made?
What is the sliding clamp for, and why does the system need a clamp loader?
Why does replication need a helicase and topoisomerases, ahead of the replication fork?
What is different between bacterial (e.g. E. coli) and eukaryote DNA replication?
the Crick and Watson model for DNA helix was correct ( ! ! )• most of the DNA in cells is similar to
B-form double helix
2 important exceptions in living cells: replication and transcription
however
DNA bending
• DNA sequences vary in details of their structure, and in their flexibility
every base-pair step differs slightly from regular B-DNA eg. in twist and roll angles.
every DNA sequence has a unique structural 'character'
~ DNA condensation and storage: nucleosome positions, etc.~ supercoiling: DNA circles
DNA bending plays an important role (bacteria), DNA on nucleosomes (eukaryotes) ~ recognition of DNA sequences by proteins ( eg.
IHF )
how many negative charges per turn of the helix 2 strands / 10.5 per turn / 21 in total
DNA in cells is dynamic and flexible ...in general, any big deformation of the stable B-DNA
structure
is energetically unfavourable (ie. will not happen spontaneously)
the energy 'price' (DG) for bending the DNA must be 'paid' from• interactions with proteins
• supercoiling energy, etc • thermal motion/energy
DNA resists bending and twisting (or untwisting)
bending
eg. on nucleosomes
twisting
eg. in negative supercoiling
IHF (Integration Host Factor)an E. coli protein needed for
phage lambda ( l ) integration
with many other roles in transcription regulation, DNA
compaction
lambda integration : IHF + integrase ( a tyrosine

recombinase )
lamda excision: IHF + xis + integrase
DNA bending by proteins appears to be the rule rather

than the exception with the topological distortions
ranging from tens of degrees to nearly 720° in the
nucleosome core (1, 2). Bending is important in DNA
packaging and in regulating diverse cellular processes.
The Escherichia coli integration host factor (IHF),
discovered as a host protein required for lysogeny by
bacteriophage λ, plays both of these roles (3, 4). IHF is an
excellent model system for analyzing the mechanism by
which proteins bend DNA by virtue of its cellular
functions, sequence specificity, tight binding, and robust
protein-induced DNA bending (Fig. 1 and refs. 5 and 6).
The studies of Sugimura and Crothers (7) and Ansari and
coworkers (8) in a recent issue of PNAS directly follow the
time evolution of DNA bending upon IHF binding to show
that bending sequentially follows protein binding.
IHF is a member of a family of Eubacterial proteins that

dramatically bend DNA. Although some family members
are sequence nonspecific in their binding, IHF binds to
specific DNA sequences with nanomolar affinity. DNA
structure is important in site selection by all of the family
members: DNA bends, nicks, and kinks facilitate binding
(6, 10). IHF is a 22-kDa heterodimer of two similar subunits
IHF, a well-studied architectural DNA bending
that fold around one another to form a single, compact
protein
"body" from which two long β ribbon "arms" extend (Fig. 1).
The DNA and protein engage in a mutual embrace: the
DNA wraps around the body of the protein whose arms in
turn wrap around the minor groove of the DNA.
The IHF-induced DNA bend appears to be stabilized by
two mechanisms. First, IHF lines the inside face of the DNA
with positive charge, allowing bending by mutual
repulsion of the phosphates on the outside face of the
DNA. Second, the β ribbon arms reach around to the
outside of the complex, where the prolines at their tips
intercalate between base pairs, compensating for the
disruption in stacking present in the tight bend. The bound
DNA entails three relatively straight segments separated
by the two large kinks where the proline residues are
intercalated
The "U-turn" bend introduced by IHF brings sequences

distant along the DNA duplex into spatial proximity (Fig. 1).
This geometry consolidates signals in the primary
sequence and could facilitate the cooperative binding of
regulatory proteins by means of an "indirect" mechanism.
Such a mechanism is observed between IHF and the
gpNu1 subunit of λ terminase, the viral DNA packaging
enzyme. IHF and gpNu1 bind to cos, the packaging
initiation site of λ DNA (Fig. 2). IHF-induced bending at the
I1 site juxtaposes the two gpNu1 half-sites, facilitating
binding. Conversely, binding of a gpNu1 dimer at R3 and
R2 introduces a strong bend into the duplex at I1,
facilitating IHF binding (Fig. 2and ref. 9). Cooperative
binding of IHF and gpNu1 is thus mediated exclusively by
each protein providing a "prebent" architecture without
direct interaction. This example illustrates a general
mechanism for the assembly of multicomponent
nucleoprotein complexes
example: how IHF helps to build complex

protein/DNA architectures
Slide 1
IHF protein induces a DNA bend of ~ 180o from a crystal structure of IHF bound to a specific DNA sequence
(more about sequnce specificity on day 3)

90 degrees kink: v. wide minor groove mostly roll at one
bp step
IHF bends the DNA at two ~90o kinks
u turn in dna / 2 sharp kinks /
1. reduced stacking of flat basepairs

(in B-DNA, stacking maximizes hydrophobic contacts of basepairs)
a sharp bend in DNA is energetically costly: 2. increased charge repulsion of phosphates

(in straight B-DNA, phosphates charges are all equally distant)
these energy costs would usually keep the DNA straight
1. IHF inserts a
hydrophobic wedge P=proline
between two basepairs ~90o kink
two ways that IHF stabilizes the DNA bends
2. charge neutralization: IHF surface has many

positively charged aa's
protein residues that often 'read' distinctive

features in DNA .....

contain all the information for the structure and functioning of a cell
Bacterial genomes are generally smaller and less variant in size among species when compared
with genomes of eukaryotes. Bacterial genomes can range in size anywhere from about 130
kbp[1][2] to over 14 Mbp.[3] A study that included, but was not limited to, 478 bacterial genomes,
concluded that as genome size increases, the number of genes increases at a disproportionately
slower rate in eukaryotes than in non-eukaryotes. Thus, the proportion of non-coding DNA goes
up with genome size more quickly in non-bacteria than in bacteria. This is consistent with the fact
that most eukaryotic nuclear DNA is non-gene coding, while the majority of prokaryotic, viral,
and organellar genes are coding.[4] Right now, we have genome sequences from 50 different
bacterial phyla and 11 different archaeal phyla. Second-generation sequencing has yielded many
draft genomes (close to 90% of bacterial genomes in GenBank are currently not complete); third-
generation sequencing might eventually yield a complete genome in a few hours. The genome
sequences reveal much diversity in bacteria. Analysis of over 2000 Escherichia coli genomes
reveals an E. coli core genome of about 3100 gene families and a total of about 89,000 different
gene families.[5] Genome sequences show that parasitic bacteria have 500-1200 genes, free-
living bacteria have 1500-7500 genes, and archaea have 1500-2700 genes.[6] A striking discovery
bacterial genomes by Cole et al. described massive amounts of gene decay when comparing Leprosy bacillus to
ancestral bacteria.[7] Studies have since shown that several bacteria have smaller genome sizes
than their ancestors did.[8]Over the years, researchers have proposed several theories to explain
the general trend of bacterial genome decay and the relatively small size of bacterial genomes.
Compelling evidence indicates that the apparent degradation of bacterial genomes is owed to a
deletional bias
E. coli (fixed)DNA in nucleoid: blue cell membrane: green
typical bacterial genome (eg. TB (M. tuberculosis) or E. coli

~4,000,000 bp ~1.3 mm of DNA
~1 Mbyte of information
~3,000 genes
4bp = 8bits = 1byte

TGGC 11101001

eg. Staphylococcus aureus (MRSA strain)~3 Mbp ~3,000
genes. all replicated in ~ 1 hour ( ! )
map shows ~ 0.6% of S. aureus genome ... ~ 18,000

bpproteins encoded in all 6 frames. but mostly on 1 strand
(•• why?)
all these 'files' (genes) need to be read (transcribed) most

genes are 'on' by default
... eukaryotic genomes are even bigger ...

bacterial genomes: information-rich, replicated
very quickly bacterial genome (eg. M. tuberculosis)
~4,000,000 bp ~1 mm of DNA
~1 Mbyte
~3,000 genes
human genome ~3,000,000,000 bp

~1 metre of DNA ~750 Mbytes
≤ 25,000 genes
most genes 'off' by default
digital devices store more information, take up far more

space
fibroblasts
~2 m human genome fits into
~6 μm cell nucleus(wound on nucleosomes, + further condensation..
DNA bending: essential to fit chromosomes into
cells
DNA structure on nucleosome core
(eukaryotes only!)
DNA bends fairly smoothly:~ 1.8 turns (700o) in ~ 140 bp DNA structure: B-like helix

The Structure of DNA

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DNA 4Y - The structure of DNA

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DNA 4Y - The structure of DNA

- stores 'genetic information' in strings of four 'characters' (A, C, G, T)

- The information on DNA must be:

- copied (replication)archived (chromatin etc.)

What is DNA for? - maintained/repaired

DNA- Deoxyribonucleic acid

it is deoxy because - which contains one less oxygen-containing hydroxyl group

RNA can be an enzyme unlike DNA

linear code of strings of info of those 4 characters

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

"The uniqueness of a given DNA structure lies solely in the

each of these strands has the chemical structure// each

sequence is bases is what's unique

The building blocks of DNA

- bases are flat ( double bonded )

numbering of bases = purines

DNA 4Y - The structure of DNA

A 'chemist's' drawing of a nucleotide, showing the

to distinguish the atoms from the base, there's a priming

5'C - not part of the ring that's attached to a phosphate

3'c - important positions / chain carries on from this point

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

Cytosine and Thymine

conformation at glycosidic bond syn-pyrimidine (T)

All the nucleotides in A and B-DNA are anti-

DNA 4Y - The structure of DNA

Purines: Adenine and Guanine

Pyrimidines: Thymine, Cytosine, and Uracil

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

DNA 4Y - The structure of DNA

The two Watson-Crick basepairs have very similar sizes

"major groove" and "minor groove" indicate where the

hydrogen atoms don't show up

DNA 4Y - The structure of DNA

The flat surfaces of the basepairs (i.e. their 'top'and

they'll come as close as together until van Der waals

Base stacking is a common arrangement of nucleobases

DNA double helix: In double-stranded DNA, bases from

DNA 4Y - The structure of DNA

ThebasisofavanderWaalsinteractionis that the distribution of electronic charge around an atom

DNA 4Y - The structure of DNA

A final fundamental interactioncalled the hydrophobic effect is a manifestation of the proper-ties

DNA 4Y - The structure of DNA

What holds the DNA in a double helix structure?

Building the double helix

DNA 4Y - The structure of DNA