Download A-DNA

Molecular Biology II
BCH 446
Dr. Amina R ELGezeery
Biochemistry Dept
King Saud University
Continuous Assessment Tests (CAT)
• • Two Tests --------------------------40 Marks
• • Two quiz --------------------------10 Marks
• • Final----------------------------------50 Marks
Dates for CAT :
– 1st CAT: … Sunday 23 Dhu-Al-Qadah 1431
– 2nd CAT: … Tuesday 8 Muharram 1432
• Time: 1.00-2.00
• Lecture Room: B 8 / R 686
Ref. Books
• • Lehninger: Pronciples of Biochemistry by
DL. Nelson and MI. Cox .
• From genes to genomes . Dale J W and von
Schantz M .
Outline of lectures 1&2
• Introduction to molecular biology :
• DNA as a genetic material.
• DNA Structure and characters
Molecular Biology
• Molecular biology is the study of gene
structure and functions at the molecular level
to understand the molecular basis of
hereditary , genetic variation, and the
expression patterns of genes .
• The molecular biology field overlaps with
other areas , particularly genetic ,
biochemistry ,bacteriology and cell biology .
What is the Molecule of Hereditary ?
Proteins?
DNA?
RNA?
•Griffith’s Experiment
•Avery and Macleod Experiment
•Hersey and ChaseExperiment
• DNA was discovered in 1869 by Friedrich Miescher as a new,
acidic, phosphorus containing substance made up of very
large molecules that he named “nuclein”, but its biological
role was not recognized.
In 1889 Richard Altmann introduced the term “nucleic acid”.
By 1900 the purine and pyrimidine bases were known. Twenty
years later, the two kinds of nucleic acids, RNA and DNA, were
distinguished .
• In (1928)and (1944) it was indicated that
DNA . could be the carrier of genetic
information .
Griffith’s Experiment
Griffith’s
Experiment
The Transforming Principle
Experiment of Avery, Macleod and McCarty (1944)
Avery, MacLeod, and McCarty (1944) , elucidated the chemical
basis of the transforming principle. From cultures of an S strain (1)
they produced an extract of lysed cells (cell-free extract) (2). After
all its proteins, lipids, and polysaccharides had been removed, the
extract still retained the ability to transform pneumococci of the R
strain to pneumococci of the S strain (transforming principl) (3) .
Avery and co-workers determined that this was attributed to the
DNA alone. Thus, the DNA must contain the corresponding
genetic information.
This explained Griffith’s observation. Heat inactivation had left the DNA of the
bacterial chromosomes intact. The section of the chromosome with the gene
responsible for capsule formation (S gene) could be released from the destroyed S
cells and be taken up by some R cells in subsequent cultures. After the S gene was
incorporated into its DNA, an R cell was transformed into an S cell (4).
The Hershey and Chase Experiment
The final evidence that DNA, and no other molecule, transmits
genetic information was provided by Hershey and Chase in
1952.
They labeled the capsular protein of bacteriophages with
radioactive sulfur (35S) and the DNA with radioactive
phosphorus (32P).
When bacteria were infected with the labeled bacteriophage,
only 32P (DNA) entered the cells, and not the 35S (capsular
protein).
The subsequent formation of new, complete phage particles in
the cell proved that DNA was the exclusive carrier of the
genetic information needed to form new phage particles,
including their capsular protein.
DNA is the carrier of genetic informations’
‘
Page 84
Diagram of T2 bacteriophage injecting its DNA
into an E. coli cell.
DNA as the Carrier of Genetic
Material
• Any substance which form the heriatable material must
fulfill some essential requirements and DNA was found
to fulfill them .
• 1- It is stable .
• 2- It is able to carry and transcribe information which
are required to control the processes which give the
organism its specificity .( transcription )
• 3- It is capable of replicating exactly, so that the genetic
determinants are transmitted down from cell to cell
and from generation to generation unchanged .
• 4- It is able to mutate .
DNA
–Location
–Structure
–Biosynthesis
–Function
–Synthesis of RNA
–Gene Expression
•
Genome: entire complement of DNA molecules of
each organism
Overall function of genome:
-Control the generation of molecules (mostly RNA &
proteins) that will regulate the cell function and
structure .
- Transfer the genetic information from cell to cell (
cell division ) and from generation to generation
without change .
DNA :
Double helix
Stores genetic code as
a linear sequence of
bases
≈ 20 Å in diameter
Human genome ≈ 3.3 x
109 bp
≈ 25,000 genes
The DNA Double Helix

General structural features

The double-bonded structure is stabilized by

1. Hydrogen bonding between complementary bases



A bonded to T by two hydrogen bonds
C bonded to G by three hydrogen bonds
2. Base stacking

Within the DNA, the bases are oriented so that the flattened
regions are facing each other
The DNA Double Helix

General structural features

There are two asymmetrical grooves on the
outside of the helix

1. Major groove

2. Minor groove

Certain proteins can bind within these grooves

They can thus interact with a particular sequence of bases
The DNA Double Helix

General structural features



Two strands are twisted together around a
common axis
There are 10 bases per complete twist
The two strands are antiparallel


One runs in the 5’ to 3’ direction and the other 3’ to 5’
The helix is right-handed

As it spirals away from you, the helix turns in a
clockwise direction
A pairs with T (2 H-bonds)
G pairs with C (3 H-bonds)
Schematic model
Space-filling model
Coding strand 5’→ 3’ .
Non-coding strand 3’ → 5’ .
“Chargaff’s rules”
1. The base composition of DNA generally varies from one species to
another.
2. DNA specimens isolated from different tissues of the same species
have the same base composition.
3. The base composition of DNA in a given species does not change
with an organism’s age,nutritional state, or changing environment .
4. In all cellular DNAs, regardless of the species, the number of
adenosine residues is equal to the number of thymidine residues
(that is, A T) ,and the number of guanosine residues is equal to
the number of cytidine residues (G C).
From these relationships it follows that the sum of the purine
residues equals the sum of the pyrimidine
residues; that is, A + G= T + C.
Nucleic acid structure can be described in terms of hierarchical
levels of complexity as primary, secondary and tertiary
structures.
-The primary structure of a nucleic acid is its covalent
structure and nucleotide sequence.
-Any regular, stable structure taken up by some or all of
the nucleotides in a nucleic acid can be referred to as
secondary structure ( eg .Double helex ) .
-The complex folding of large chromosomes
within eukaryotic chromatin and bacterial nucleoids is
generally considered tertiary structure.
DNA wound
around histone
proteins
•Gives Maximum Absorption at OD 260.
•Denaturation: dsDNA ssDNA
•Melting Temperature (Tm): Temperature at which 50% of
the dsDNA is changed to ssDNA. Depends on GC content.
•Hyperchromiceffect: on denaturationthe OD260 increases.
•Hypochromic effect: on renaturation the OD260decreases.
•OD260/OD280 ratiois around 2 for pure DNA sample.
•Extinction Co-efficientof DNA: 20 for 1mg/ml DNA( The
Beer-Lambert Law can be used for calculation of DNA
concentration: A= ΕxCxl ).
• Why the absorbance of ssDNA is higher than
that of dsDNA ?
• Is DNA denaturated in vivo ? Explain
Different Structural Forms Of
Nuclear DNA

The DNA double helix can form different
types of secondary structure

The predominant form found in living cells is
B-DNA

However, under certain in vitro conditions,
A-DNA and Z-DNA double helices can form

B-DNA
* The B form is the most stable structure for a randomsequence DNA molecule under physiological conditions and is
therefore the standard point of reference in any study of the
properties of DNA.



Right-handed helix
10 bp per turn
Most stable form .

A-DNA




Right-handed helix
11 bp per turn
Occurs under conditions of low humidity
Little evidence to suggest that it is biologically important
-The reagents used to promote crystallization
of DNA tend to dehydrate it, and thus most short
DNA molecules tend to crystallize in the A form.
Whether A-DNA occurs in cells is uncertain .

Z-DNA
Left-handed helix
12 bp per turn
The DNA backbone takes on a zigzag appearance .
The major groove is barely apparent in Z-DNA, and the
minor groove is narrow and deep.
There is evidence for some short stretches (tracts) of ZDNA


Its formation is favored by



GG-rich sequences, at high salt concentrations
Cytosine methylation, at low salt concentrations
Evidence from yeast suggests that it may play
a role in transcription and recombination
Right Handed B & A forms
Left Handed Z form
Bases
substantially
tilted relative
to the central
axis
Bases
substantially
tilted relative
to the central
axis
Bases relatively
perpendicular to the
central axis
Sugar-phosphate
backbone follows a
zigzag pattern
Certain DNA Sequences Adopt Unusual
Structures
A number of sequence-dependent structural variations have
been detected within larger chromosome that may affect the
function and metabolism of the DNA segments
1- Bends occur in the DNA helix wherever four or more
adenosine residues appear sequentially in one strand.
Six adenosines in a row produce a bend of about 18.
The bending observed with this and other sequences may
be important in the binding of some proteins to DNA.
2- palindrome Sequence :
A palindrome is a word, phrase, or
sentence that is spelled identically
read either forward or backward ;eg.
ROTATOR .
The term is applied to regions of DNA
with inverted repeats of base
sequence having twofold symmetry
over two strands of DNA .
Such sequences are selfcomplementary within each strand
and therefore have the potential to
form hairpin or cruciform(crossshaped) structures .
Inverted repeat can lead to loop formation
Complementary regions
Held together by
hydrogen bonds
Noncomplementary regions
Have bases projecting away
from double stranded regions
Also called
hair-pin
Holliday junction
DNA cruciform
Sequences of these types are found in virtually every large
DNA molecule and can encompass a few base pairs or
thousands.
The extent to which palindromes occur as cruciforms in cells
is not known, although some cruciform structures have been
demonstrated in vivo in E.coli.
Self-complementary sequences cause isolated single strands
of DNA (or RNA) in solution to fold into complex structures
containing multiple hairpins.
3- Mirror repeat sequence :
When the inverted repeat occurs within each
individual strand of the DNA, the sequence is called a
mirror repeat.
Mirror repeats do not have complementary sequences
within the same strand and cannot form hairpin or
cruciform structures.
4- H-DNA :
It is found in polypyrimidine or polypurine tract that also incorporate a
mirror repeat.
A simple example is a long stretch of alternating T and C residues .
The H-DNA structure features the triple-stranded. Two of the three strands in
the H-DNA triple helix contain pyrimidines and the third contains purines .
(a) A sequence of alternating T and
C residues
(b) These sequences form an unusual
structure in which the strands in one half of
the mirror repeat are separated and the
pyrimidine containing strand (alternating T
and C residues) folds back on the other
half of the repeat to form a triple helix. The
purine strand (alternating A and G residues) is
left unpaired. This structure produces a sharp
bend in the DNA.
H DNA
In the DNA of living cells, sites recognized by many
sequence-specific DNA-binding proteins are arranged as
palindromes, and polypyrimidine or polypurine
sequences that can form triple helices or even H-DNA
,are found within regions involved in the regulation of
expression of some eukaryotic genes.
Synthetic DNA strands designed to pair with these
sequences to form triplex DNA could disrupt gene
expression.
This approach to controlling cellular metabolism is of
growing commercial interest for its potential application
in medicine and agriculture.
DNA Can Form a Triple Helix

In the late 1950s, Alexander Rich et al discovered
triplex DNA


It was formed in vitro using DNA pieces that were made
synthetically
In the 1980s, it was discovered that natural doublestranded DNA can join with a synthetic strand of
DNA to form triplex DNA

The synthetic strand binds to the major groove of the
naturally-occurring double-stranded DNA
- A cytidine residue (if protonated) can pair with the
guanosine residue of a GC nucleotide pair, and a thymidine
can pair with the adenosine of an AT pair .
- The N-7, O6, and N6 of purines, the atoms that participate in
the hydrogen bonding of triplex DNA,are often referred to as
Hoogsteen positions, and the non-Watson-Crick pairing is
called Hoogsteen pairing .
- The triplexes are most stable at low pH .
- The triplexes form most readily within long sequences
containing only pyrimidines or only purines in a given strand.
- Some triplex DNAs contain two pyrimidine strands and one
purine strand; others contain two purine strands and
one pyrimidine strand.

T binds to an
AT pair in
biological DNA


C binds to a
CG pair in
biological DNA
Triplex DNA formation
is sequence specific
The pairing rules are
Triplex DNA has been
implicated in several
cellular processes


Replication,
transcription,
recombination
Cellular proteins that
specifically recognize
triplex DNA have been
recently discovered
61
Bond
representation of triplex DNA. This view is down the long axis. The “third” strand is colored.
62
Four DNA strands can also pair to form a tetraplex
(quadruplex), but this occurs readily only for DNA
sequences with a very high proportion of guanosine
residues The guanosine tetraplex, or G tetraplex, is
quite stable over a wide range of conditions.
triplex and even quadruplex pairing can take place.
These structures are critical for the proper
replication of chromosomal DNA and repair of
damaged DNA.
Also there is a tendency for many of these unusual
structures to appear at sites where important events
in DNA metabolism (replication, recombination,
transcription) are initiated or regulated
64
Quadruplex DNA