Mrs. Ofelia Solano Saludar: Department of Natural Sciences University of St. La Salle Bacolod City

Mrs.
OFELIA SOLANO SALUDAR

Department of Natural Sciences
University of St. La Salle
Bacolod City
DNA is the stuff our genes are made of…
 The organization of total sum of genetic information (or
genome) of an organism is in the form of double-stranded
DNA, except that viruses may have single-stranded DNA,
single-stranded RNA or double-stranded RNA genomes.
 In many viruses and prokaryotes, the genome is a single linear
or circular molecule.
 In eukaryotes, the nuclear genome consists of linear
chromosomes (usually as a diploid set) and the mitochondrial
and chloroplast (in plants) genomes are small circular DNA
molecules.
 In 1952, Watson and Crick proposed that DNA is a double helix
which is known to have alternative forms.
Models of known DNA structures. (a) The B form of DNA has ≈10.5
base pairs per helical turn. Adjacent stacked base pairs are 0.36 nm apart. (b)
The more compact A form of DNA has 11 base pairs per turn and exhibits a
large tilt of the base pairs with respect to the helix axis. (c) Z DNA is a left-
handed double helix.
Many DNA Molecules Are Circular
DNA supercoils can be removed by cleavage
of one strand. (a) EM of SV40 viral DNA.
When the SV40 circular DNA is isolated, the
DNA duplex is underwound and assumes the
supercoiled configuration. (b) If a supercoiled
DNA is nicked, the strands can rewind, leading
to loss of a supercoil. Topoisomerase I
catalyzes this reaction and also reseals the
broken ends. All the supercoils in isolated
SV40 DNA can be removed by the sequential
action of this enzyme, producing the relaxed-
circle conformation.
In general,
genome size
increases
with the
complexity
of organism.
Chemical stability of DNA depends on:
1. Base stacking interactions - hydrophobic interactions resulting from the individual
base pairs’ stacking on top of each other in the nonpolar interior of the double helix,
electrostatic forces between nearest-neighbor base pairs, and from van der Waals
forces between the bases.
2. Deoxyribose sugar- this is less reactive because of C-H bonds. Consequently, DNA
has smaller grooves which hinder attachment of damaging enzymes that attack DNA.
3. Hydrogen bonds- the sum of all the H-bonds between the paired bases leads to a
stabilizing "zipper effect.“
4. Protective "twisting" of the DNA helix and flexibility of the two strands -
although the bases cannot rotate freely about the axis of their bonds with each other,
they are able to rotate around their bonds with the sugars. This area of rotation is like
a joint on a human arm. In DNA, there are several flexible bonds:
a. bonds between oxygen and phosphorus in phosphate groups
b. bonds linking the phosphate groups to the sugar rings
c. bonds which link the sugar rings to the aromatic bases
5. Interaction with histones- chains of DNA become even more stable, as they
entwine with histones. DNA ribbons coil around histones for protection, like a string
on a spool.
Prokaryotes package DNA in
bacterial chromosomes and
plasmids.
 The bacterial chromosome is
localized in the nucleoid region of
the cell (no nucleus) and is looped
into negative coils.
 The loops are 50,000 to 100,000
bps in length (similar to eukaryotic
chromosomes) which are held in
place by RNA and small basic
(histone-like) proteins.
 Plasmids, small negatively
supercoiled circular DNA
molecules, carry usually non-
essential genes (often drug
resistance).
Eukaryotic DNA is packaged into chromosomes
 A chromosome is formed from a single, enormously long
DNA molecule that contains a linear array of many genes.
 The human genome contains 3.2 × 109 DNA nucleotide
pairs, divided between 22 different autosomes and 2 sex
chromosomes.
Chromosomal banding patterns and multicolor FISH are used to
analyze human anomalies
Characteristic chromosomal translocations are associated with certain
genetic disorders and specific types of cancers. In nearly all patients with
chronic myelogenous leukemia, the leukemic cells contain the Philadelphia
chromosome, a shortened chromosome 22 [der (22)], and an abnormally long
chromosome 9 [der (9)]. These result from a translocation between normal
chromosomes 9 and 22.
Comparative studies reveal that human genomes contain genes in the same order
as another mammal, a feature called conserved synteny. Using chromosome
banding/ painting, the phylogenetic history of our own chromosomes maybe
reconstructed by comparing them with those from other mammals.
DNA in a eukaryotic chromosome contains genes, many
replication origins, one centromere, and two telomeres. These
sequences ensure that the chromosome can be replicated efficiently
and passed on to daughter cells.
If each nucleotide pair is
drawn as 1 mm as in (A),
then the human genome
would extend 3200 km
(approximately 2000 miles),
far enough to stretch across
the center of Africa, the site
of our human origins (red
line in B). At this scale, there
would be, on average, a
protein-coding gene every
300 m. An average gene
would extend for 30 m, but
the coding sequences in this
gene would add up to only
just over a meter.
Human DNA, if fully extended, would have a total
length of 1.7 m. If you unwrap all the DNA you have
in all your cells, you could reach the moon ...6000
times!
 Interphase chromosomes contain both condensed and more
extended forms of chromatin
 Constitutive heterochromatin - found in the centromere,
nucleolar organizers (found in human chromosomes
13,14,15,21, and 22), repetitive or satellite DNA
 Facultative heterochromatin- one of the homologues
become heterochromatic, e.g. X chromosome becomes Barr
body
 Euchromatin – loosely packed, actively transcribed regions
of the chromosome.
 Chromatin structure is dynamic: by temporarily altering its
structure by using chromatin remodeling complexes and
enzymes that modify histone tail, the cell can ensure that
proteins involved in gene expression, replication, and repair
have rapid, localized access to the necessary DNA sequences.
Chromatin remodeling
complexes alter
nucleosome structure.
Different
chromatin remodeling
complexes disrupt
and re-form
nucleosomes. The
same complex might
catalyze both reactions.
The DNA-binding
proteins could be
involved in gene
expression, DNA
replication, or DNA
repair.
The pattern of modification of histone tails may dictate how a stretch of
chromatin is treated by the cell. Each histone can be modified by the covalent
attachment of different molecules. Histone H3, for example, can receive an
acetyl group (Ac), a methyl group (Me), or a phosphate (P). Note that some
positions (e.g., lysine 9 and 27) can be modified in more than one way.
Different combinations of histone tail modifications may constitute a type
of “histone code.” Each marking conveys a specific meaning to the stretch
of chromatin on which it occurs. Only a few of the meanings of the
modifications are known.
CENTROMERE  Contains alpha
satellite
sequences
(5,000-15,000
copies of 171
base pair
sequences).
 Position of
centromere
 P and q arms
Within the centromere region, the
actual location where the
attachment of chromosomes to
spindle fibers occurs is called the
kinetochore and is composed of
both DNA and a protein called
CEN DNA. It can be moved
from one chromosome to another
and still provide the chromosome
with the ability to segregate.
CEN DNA consists of several
sub-domains, CDE-I, CDE-II
and CDE-III. Additional
analyses of the DNA and protein
components of the centromere
are necessary to fully understand
the mechanics of chromosome
segregation.
TELOMERE
 Telomeres are non-sticky regions that
prevent DNAse from degrading ends
and fusion of chromosomes.
 They facilitate replication without loss
of material.
 Most species have telomeric 3’G
overhangs that form G-quartets
(Hoogstein base-pairing)
 Contain tandem repeats which are
highly conserved (TTAGGGG in
man)
 These 500-3,000 repeats in normal
cells shorten with age
Telomere
replication
The 3′ end of the
parental DNA strand is
extended by RNA-
templated DNA
synthesis; this allows
the incomplete daughter
DNA strand that is
paired with it to be
extended in its 5′
direction. This strand is
presumed to be
completed by DNA
polymerase α, which
carries a DNA primase
as one of its subunits.
Telomerase adds
TTGGGG to end of
lagging strand, which
then forms a hairpin
turn. The end of the
lagging strand has a
primer with a free 3’-
OH end that
polymerase can
extend from. This
primer is later
removed.
How shortened telomeres
may lead to chromosomal
instability and cancer
Most human cells lack
telomerase. In normal cells
that still produce functional
p53 and have their cell-cycle
checkpoints intact, this
triggers cell death. But a cell
that has acquired a p53
mutation may ignore this
signal and cause massive
chromosomal damage. Some
cells reactivate telomerase,
which restores enough
chromosomal stability for cell
survival. These damaged cells
can then go on to accumulate
the additional mutations
needed to produce a cancer.
 The mitochondria and chloroplasts also have a DNA genome (or
chromosome). These resemble procaryotic genomes (likely due to the
endosymbiotic origin of these organelles) but are much smaller.
 The mitochondrial genome varies in size among eukaryotes (mammals
=16.5 kb & 37 genes, yeast and plants are greater than 5X this).
 Chloroplasts are ~120
kb and have ~120 genes.
DNA in ORGANELLES
DNA Can Undergo Reversible Strand Separation
Denaturation or “melting,”(unwinding and separation of DNA
strands), can be induced by increasing the temperature of a
solution of DNA.
 Denaturation and renaturation of DNA are the basis of nucleic
acid hybridization
 Loss of the multiple weak interactions holding the strands
together along the entire length of the DNA molecules lead to
an abrupt change in the absorption of ultraviolet (UV) light.
 The melting temperature (Tm ) at which DNA strands will
separate depends on several factors:
a. When the ion concentration is low, shielding of negatively
charged phosphate groups in the two strands by positively
charged ions is decreased, thus increasing the repulsive
forces between the strands and reducing the Tm.
b. A greater proportion of G-C pairs require higher temperatures to
denature.
c. pH extremes denature DNA at low temperature. At low pH, the bases
become positively charged, repelling each other. At high pH, the
bases become negatively charged, again repelling each other because
of the similar charge.
d. Agents that destabilize hydrogen bonds, such as formamide or urea,
also lower the Tm.
Through the analysis of DNA renaturation studies, the large sizes of
eukaryotic genomes reveal large amounts of repeated DNA. These undergo a
complex pattern of re-annealing which reveals a large amount of repeated
DNA sequences (fast annealing) and unique, non-repeated DNA (slow
annealing).

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