Structural Organization of DNA

Academic Script

Introduction

DNA is vital to the field of genetics and its organization has been an intriguing concept for
the scientists from ages. The living cells exist by virtue of some macromolecules like the
proteins, carbohydrates,
lipids, nucleic acid etc. The cell is
the repository of information and
instructions made of DNA in unique
and different sequences. It is
the genes that store the information.

DNA is tied together with proteins to

form chromatin fibers that further form
the chromosomes. This association
of eukaryotic DNA allows DNA to be
accurately replicated and sorted into
daughter cells without much error and
intertwinement during cell division.

Circular DNA molecules called

plasmids are found in prokaryotic cells,
which are stored within the cell's
cytoplasm. On the contrary, eukaryotic chromosomes have several levels of organization.
Eukaryotic DNA coil around histone proteins to form histone-DNA complexes called
nucleosomes, which are organized into large, coiled loops held together by interlining
proteins.

In this chapter, we establish the organization of DNA and its dynamics in a certain
organism like the viruses and eukaryotes and understand how they affect the function of
the cell. We will also dwell in some interesting concepts of split genes, DNA palindromes
and transposons.
DNA Organization
in Viruses

The nucleic acid which

encodes the genetic
information of the virus has a
varied composition &
structure. Therefore, the
virus genomes (i.e. the entire
gene structure) is more varied
compared to the entire
bacterial, plant or animal
kingdoms.

The nucleic acid comprising

the genome may be single-stranded or double-stranded, & in a linear, circular or
segmented configuration. Single-stranded virus genomes may be:

arity (nucleotide sequence) as mRNA

-)sense
-a mixture of the two
Virus genomes range in size from approximately 3,200 nucleotides (nt) like that of
Hepadnaviruses to approximately 1.2 million base pairs like that of Mimivirus.
Virus genomes may contain their genetic information encoded in either DNA or RNA,
which is in contrary to the genomes of all cells, which are composed of DNA. Since viruses
are obligate intracellular parasites, which mean, they will be able to replicate in the
appropriate host cells, the genome must contain information encoded in a form which can
be recognised & decoded by the particular type of cell parasitized. Thus, the host organism
should match the genetic code employed by the virus. Similarly, the control signals which
direct the
expression of
virus genes
must be fitting
to the host.

The biology of
their genomes
closely
resembles their
host cells in
terms of the
DNA viruses of
eukaryotes.

Some DNA virus genomes are coupled with cellular histones to form a chromatin-
like structure within the virus particle.

Kates in 1970 was the first to observe the phenomenon that Vaccinia virus mRNAs
were found to be polyadenylated at their 3' ends.

Sharp in 1977 was the first to discover split genes containing non-coding introns,
protein coding exons & spliced mRNAs in adenoviruses.
The new techniques of molecular biology have had a major influence in concentrating
much attention on the virus genome. The estimation of the size of nucleic acid molecules
can be done by direct analysis by electron microscopy, which should be calibrated with
known standards.

The most important single technique has been gel electrophoresis. It is most common to
use agarose gels to separate large nucleic acid molecules (several megabases or
kilobases) & polyacrylamide gel electrophoresis (PAGE) to separate smaller pieces (a few
hundred bp down to a few nucleotides).

The sequencing of nucleotide is dependent on the ability to separate molecules which

differ from each other by only one nucleotide in size.
One of the major advantages due to the relative simplicity of virus genomes (compared
with even the simplest cell) is the ability to 'rescue' infectious virus from purified or cloned
nucleic acids.
Transfection is
the infection of
cells caused by
nucleic acid
alone.

Virus genomes
which include (+)
sense RNA (i.e.
the same polarity
as mRNA) are
infectious when
the purified vRNA
is applied to cells without any virus proteins. This is because (+) sense vRNA is essentially
mRNA & the first event in a normally-infected cell is to translate the vRNA to make the
virus proteins responsible for genome replication. In this case, direct introduction of RNA
into cells merely circumvents the earliest stages of the repeated cycle.

Mostly, virus genomes which are composed of double-stranded DNA are also infectious.
The virus genome must first be transcribed by host polymerases to produce mRNA, thus
making the events more complex. The virus can be rescued from cloned genomes,
including those which have been manipulated in vitro, by using the above mentioned
techniques.

Split Genes

The discovery of the nature of the genes of complex multi-cellular organisms (eukaryotes)
happened in 1977. It was found that the genes contain intervening sequences that are
removed from the RNA transcript shortly after transcription. These excised sequences
were named introns and the gene fragments they separate exons by Gilbert in 1978. Split
genes are also known as the interrupted genes.

The discovery of introns provided a major difference between the genes of prokaryotes
(bacteria) and eukaryotes, and raised the presence of fundamental functional distinction
between these groups in processes such as genetic regulation.
To summarize,
the intervening
sequences in split
genes and
transcript
processing are
called introns and
the fragments
they separate are
called exons.
Splicing is the process of intron removal and the unprocessed transcript is called a pre-
mRNA. Additionally, the removal of introns, the mature eukaryote mRNA transcript has
been capped at the 5' end (to prevent degradation), and the 3' end has been ploy-
adenylated to give a tail of adenine nucleotides hundreds of nucleotides long.

Bacterial DNA Organization

The initial perception about the bacterial DNA structure was that they are circular in nature
and there were ample amount of studies and evidence presented to justify the theory.
However, in reality it is the linear chromosomes found in eukaryotic cells. Bacterial
plasmids were also shown to be circular. In fact, the experiments were so beautiful and the
evidence was so convincing that the idea that bacterial chromosomes are circular and
eukaryotic chromosomes are linear was quickly accepted as a definitive distinction
between prokaryotic and eukaryotic cells. However, like most other distinctions between
prokaryotic and eukaryotic cells, it is now clear that this theory is incorrect. It is now
established that not all bacteria have a single circular chromosome: some bacteria have
multiple circular chromosomes, and many bacteria have linear chromosomes and linear
plasmids, which brings in more variation to the organization of the DNA in bacteria.

Experimental evidence for multiple chromosomes and linear chromosomes initially came
from studies using pulsed field gel electrophoresis (PFGE), an approach that uses
alternating electric fields to separate large DNA molecules on an agarose gel.
Subsequently genome sequencing projects have added to the list of bacteria with multiple
or linear chromosomes.

The studies on Rhodobacter sphaeroides provided THE first convincing evidence that
some bacteria have multiple chromosomes. Both molecular and genetic studies clearly
demonstrated that sphaeroides has two large circular chromosomes. Genes encoding
rRNAs and tRNAs required for translation and metabolic enzymes are distributed between
the two chromosomes. Multiple chromosomes have also been found in many other
bacteria, including Agrobacterium tumefaciens, Rhizobium, Brucella, Paracoccus
denitrificans, Ochrobactrum anthropi, Leptospira interrogans, Burkholderia, Vibrio
cholerae, Deinococcus radiodurans and many others from diverse groups of bacteria.
In addition, some bacteria have linear chromosomes. Borrelia3 have linear chromosomes
and most strains contain both linear and circular plasmids; most of the bacteria in the
genus Streptomyces4 have linear chromosomes and plasmids and some have circular
plasmids as well. Additionally, there may be a dynamic equilibrium between linear and
circular forms of a DNA molecule. Studies with some evidence prove that linearization may
be due to integration of a linear phage genome into the circular DNA molecule.

The ends of linear DNA molecules (called telomeres) create two problems that do not
apply to circular DNA molecules. Firstly, the sensitivity to degradation of the free double-
stranded DNA ends by intracellular nucleases brings about a strong need of a mechanism
to protect the ends. Secondly, there has to be a special mechanism for DNA replication for
the ends of linear DNA molecules. These problems are solved by features of the
telomeres. Two different types of telomeres have been found in bacteria: hairpin telomeres
and invertron
telomeres.

There are
examples of
linear DNA
molecules in
bacteria that are
protected by both
types of telomeres: palindromic hairpin loops are protected by the lack of free double-
stranded ends, and invertron telomeres are protected by proteins that bind to the 5'-ends.
Both of these mechanisms are also used by some phage, eukaryotic viruses, and
eukaryotic plasmids.

The two types of telomeres also solve the problem of DNA replication differently. Invertron
telomeres have a protein covalently attached to the 5' ends of the DNA molecule. DNA
polymerase interacts with the TP at the telomere and catalyzes the formation of a covalent
bond between the TP and a dNTP. The dNTP bound to the TP has a free 3'-OH group
which acts as the primer for chain elongation. Replication of hairpin telomeres is still to be
researched for a clear understanding. It seems that multiple hairpin sequences can pair to
form concatemers that are replication intermediates.

It is vital to note that it is just beginning to become clear that the similarity of many
processes once thought to be completely different between bacteria and eukaryotes, partly
because of better tools for studying these processes and partly because most of the earlier
studies focused on relatively few types of bacteria. The more studies focus on a wider
diversity of bacteria, phages, and plasmids, the more obvious it becomes that E. coli is an
excellent model for dissecting broad features of molecular and cell biology, but not all
bacteria do everything the same way. Furthermore, it is a recent approach to study the
molecular genetics of the Archae, and what has been learnt so far suggests that this
diverse group of prokaryotes share even more common features with the eukaryotes.
The circular genomes of mitochondrial and chloroplast are a noteworthy exemption to the
rule that eukaryotic chromosomes are linear. However, there is a comfortable analogy with
the dichotomy that eukaryotic chromosomes are linear and bacterial chromosomes are
circular because these organelles seem to have evolved from entrapped bacteria.

For example, linear DNA was precipitated in the most commonly used procedures for
purifying bacterial plasmids, and the procedures for purifying chromosomal DNA relied
upon the differential binding of ethidium bromide to "sheared DNA fragments" compared to
circular DNA.

All known DNA polymerases require a pre-existing primer for initiation of DNA replication.
The primer is usually a short RNA molecule with a free 3'-OH group that can be extended
by DNA polymerase. If a linear DNA molecule was primed at one end, DNA synthesis
could continue to the other end. However, once the primer is removed, the DNA matching
the primer could not be replicated.

Transposons

Transposons, or “Jumping genes”

or Transposable elements (TEs),
are DNA sequences
that move from one location on the
genome to another. Around more
than fifty years ago, these elements
were first identified by geneticist
Barbara McClintock of Cold Spring
Harbor Laboratory in New York.
Though, the discovery
received initial scepticism from
the biologists. Research
across the following decades proved
that not only do TEs "jump," but they
are also found in almost all
organisms (both prokaryotes and
eukaryotes) and typically in large
numbers. For example, TEs
make up approximately 50% of the human genome and up to 90% of the maize genome.
Most transposons eventually become inactive and no longer move.
The functions of transposons are not stated clearly. They have often been referenced as
“junk” DNA because they appear to serve little or no purpose or as “selfish” DNA
because they serve only to copy and magnify themselves within genomes. Rarely, but in a
few instances, transposons are associated with genetic mutations or chromosomal
rearrangements that cause disease in humans.

The disease typically arises from the inclusion of transposons into particular regions of
genes that are involved in regulating gene activity.

Types of Transposons
Today, scientists know that there are many different types of TEs, as well as a number of
ways to categorize them. One of the more popular methods of segregation is between
those TEs that require reverse transcription (i.e., the transcription of RNA into DNA) in
order to transpose and those that do not. The former elements are known as
retrotransposons or class 1 TEs, whereas the latter are known as DNA transposons or
class 2 TEs. The Ac/Ds system that McClintock discovered falls in the latter category.
Different classes of transposable elements are found in the genomes of different
eukaryotic organisms.

The X-axis shows the species and a shaded vertical bar shows the relative contribution of
retrotransposons and DNA transposons as a percentage of the total number of
transposable elements. The area of the bar shaded blue represents the proportion of
retrotransposons, while the area of the bar shaded red represents the proportion of DNA
transposons. Species Sc, Sp, Hs, Mm, Dm, Ag, and Eh are between 60 and 100 percent
retrotransposons, whereas species Os, Ce, Aa, Ei, and Tv are between 70 and 100
percent DNA
transposons.
Class II Transposons
A “cut and paste” mechanism is used by Class II elements which move from one place to
another and are simple segments of DNA. The majority of these elements encode an
enzyme called transposase, which acts to cleave the ends of the transposon, releasing it
from its initial location in the genome. Transposase also cleaves target sites where the
element is to be inserted. Once the transposon is bound to its new position, gaps that are
left in the DNA sequence are filled through the synthesis of nucleotides. Class II
transposons range in length from 1,000 to as many as 40,000 base pairs.

DNA Palindromes
It is commonly perceived that the structure of the DNA is a linear double helix, however, in
a real scenario; DNA may assume many different structures, some of which have deep
influences on DNA’s biological functions. For example, palindromic DNA sequences,
which read the same in opposite directions (but on different strands), can extrude to form a
cruciform, with both strands involved or a hairpin, with only one strand involved. These
structures are then worked on by enzymes that have only slight activity on linear double-
helical DNA. There is extensive evidence that these and other noncanonical DNA
structures can have dire consequences such as initiating chromosomal translocations,
which can result in cancer or developmental defects. A palindrome, such as the famous
“Madam, I'm Adam,” reads the same in both directions. In the DNA and RNA worlds the
term means that one strand reads the same in the 5′ → 3′ direction as the complementary
strand reads in the 5′ → 3′ direction. An example is 5′-GTTAG|CTAAC-3′, where | indicates
the center of the palindrome. If sufficiently long, these sequences can extend to form a
cruciform, with a few unpaired nucleotides at the center flanked by dsDNA. When present
in ssDNA, such as during replication or transcription, the palindrome can fold into a hairpin,
equivalent to half of a cruciform. In both cases the capped end is a substrate for two
known classes of enzymes: (1) the MRN complex of eukaryotes, and its archival
equivalent MR and bacterial equivalent SbcCD; and (2) Artemis of vertebrates, involved in
cutting hairpins made by the RAG complex during V(D)J recombination, which produces
active
immunoglobulin
genes.
One of the easiest to explain palindrome is an inverted repeat (IR), in which there are
additional, unique base pairs at the center. In this case, pairing between the repeats
leaves extensive single-stranded loops of the unique base pairs at the tips of the cruciform.
The energetic cost to form this structure with single-stranded loops is large in dsDNA but
not in ssDNA. There are many instances where IRs can have a strong impact on
palindromes, apparently by folding during replication when the DNA is partly single-
stranded.

Studies have revealed that Palindromes of ∼500 base pairs (bp) or greater on human
chromosomes 11 and 22 are the sites of the most frequent recurrent human translocation
(other than Robertsonian translocations between centromeres). Reduced fertility, severe
developmental defects, including intellectual disability and cardiac defects are some of the
results of imbalance in the
palindromes.

Kurahashi recommended
that the translocation
occurs after meiotic replication,
when the chromosomes
become highly compacted for
packaging into sperm heads.
Although the molecular basis
of these debilitating translocations is
clearly palindromic DNA, the
mechanism by which
palindromes react has been most
thoroughly studied in model
organisms, especially
bacteria and yeasts.