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Transcript
Welcome to class of
DNA
Dr. Meera Kaur
Nucleic Acid Structure
• Structure of DNA:
– The most important clue to the structure of DNA
came from the work of Erwin Chargaff and his
colleagues in the late 1940s.
– The data collected from DNAs of a great many
different species led Chargaff to some important
conclusions.
These conclusions are as follows…
Chargaff’s “rules”…
• Late 1940s
• Base composition of DNA generally varies from one
species to another.
• DNA specimens isolated from different tissues of the
same species generally have the same base
composition.
• The base composition of DNA in a given species
does not change with the organism’s age, nutritional
state, or changing environment.
Chargaff’s “rules”…
• In all cellular DNAs, regardless of the species, the
number of adenine residues is equal to the number of
thymine residues (i.e., A=T), and the number of
guanine residues is equal to the number of cytosine
residue (i.e., G=C).
• From this relationships, it follows that the sum of the
purine residues equals the sum of the pyrimidine
residues, that is A+G = T+C
Chargaff’s “rules”
• Key to establishing the 3D structure of DNA
• Provide clues as to how genetic information
is encoded in DNA and passed from one
generation to the next.
DNA is a double helx
• 1950s: Rosalind Franklin and Maurice Wilkins
obtained X-ray diffraction pattern of DNA
– deduced that DNA was helical.
• The pattern also indicated that the molecule
contained two strands, a clue that was crucial to
determining the structure.
The X-ray diffraction pattern of DNA
The spots forming across in the center denote a helical
structure.The heavy bands at the top and bottom
correspond to the recurring bases.
Race was on to determine structure
• The problem was to formulate a threedimensional model of DNA molecule that
could account not only the X-ray diffraction
data, but also
– Needed to be a helical structure, also satisfy
Chargoff’s rules (A=T, C=G) and
- Satisfied other chemical properties of DNA
Watson and Crick model for the structure of DNA
• In 1953, Watson and Crick postulated that,
– It consists of two helical DNA chains coiled around the same
axis to form a right-handed double helix. The strands of DNA
comprising the double helix run in opposite direction
– The spatial relationship between the two strands creates a
major groove and a minor groove between the two strands
– The three - dimensional model of DNA structure
confirms the Chargaff’s rules
Hydrogen bonding pattern in the base pairs
defined by Watson and Crick
Schematic drawings of complementary anti parallel strand of
DNA following the pairing rules proposed by Watson and Crick
Watson-Crick model for the structure of DNA…
Watson-Crick model for the structure of DNA…
• Watson and Crick postulated that DNA is a double
helix where
- Two helical DNA strands coiled around same
axis to form right-handed double helix
- Hydrophilic backbone of alternating
sugar/phosphate units of the nucleotides are
on outside of DNA molecule exposed to water
Watson-Crick model for the structure of DNA…
• Hydrophobic bases (complementary base pairs) are
stacked neatly inside the molecule. The hydrogen
bond between the base pairs hold the double helix
together.
• Strands are anti parallel.
Watson-Crick model for the structure of DNA
Note That
• The two DNA strands in the “double helix” are anti
parallel.
• The two DNA strands are not IDENTICAL, but they
are COMPLEMENTARY.
Important features of the double helical
model of DNA
• The model explained many unusual physical
properties of DNA.
• It immediately suggested a mechanism for the
transmission of genetic information.
• It suggested a means by which DNA could be
replicated.
The central dogma
The central dogma outlines the plan for the storage
and transmission of hereditary information. Now we
know that DNA is the bearer of genetic information in
all living organisms. The entire basis of information
storage and transmission in the cell is embodied in
three steps:
REPLICATION
TRANSCRIPTION
TRANSLATION
Replication
Watson and Crick model for DNA replication
They suggested that DNA is replicated on a DNA template.
- Each strand of DNA in the double helix acts as a template – a pattern for
the synthesis of its complement. Since DNA is double-stranded,
complementary replication would produce two double-helical DNA
molecules, each containing a strand of the original DNA and a new strand
complementary to it.
- This type of replication is called semiconservative replication, which was
confirmed by M.S. Meselson and F.W. Stahl in 1957.
- In 1955, Arthur Kornberg and his associates discovered DNA
polymerase — a large protein which catalyses the formation of DNA.
- DNA strand elongates. Elongation or the chain growth is always from the
5end to the 3end of the elongating DNA molecules
Replication of DNA as suggested by Watson and Crick
Some basics
• The amino acids sequence of every protein and nucleotides
sequence of every RNA molecule in cell is specified by that cell’s
DNA.
• A segment of DNA that contains the information required for the
synthesis of a functional biological product is referred to as a
GENE.
• Ribosomal RNAs (r RNA) are the structural component of ribosome,
the large complexes carry out the synthesis of protein.
• Messenger RNAs (mRNA) are nucleic acids that carry the
information from one or a few genes to the ribosome,where the
corresponding protein can be synthesized.
• Transfer RNAs (tRNA) are adaptor molecules that faithfully translate
the information in mRNA into a specific sequence of amino acids.
Transcription
RNA is made on a DNA template
• The information contained in DNA is passed to a form of RNA called
messenger RNA (mRNA) by transcription (“rewriting”).
• The mechanics of transcription is quite similar to the mechanics of
replication because DNA is the template upon which RNA is formed.
The major difference is that—
- The enzyme is RNA polymerase instead of DNA polymerase
- Sugar units of the nucleoside used to make RNA is ribose
rather than deoxyribose, and the u (uracil) substitutes for T
(Thymidine) in RNA.
Translation
Translation involves tRNA, mRNA, ribosome and Enzymes
The synthesis of specific protein under the direction of specific gene is
complex. Proteins are the polymer of 20 different amino acids and
there are only four different nucleotide monomers in DNA. Hence,
there can not be a one-to-one relationship between the sequence
of nucleotides in the DNA molecule and the sequence of amino
acids in a protein.
The protein-coding information is read by the cells in blocks of three
nucleotides residues, or codons. Each codon specifies a different
amino acids.
The set of rules that specifies which nucleic acid codon corresponds
to which amino acid is known as genetic code.
Crick’s adaptor hypothesis
The basic principles of Translation
– A mRNA molecule is bound to a ribosome.
– The transfer RNA(tRNA) molecules bring amino acids to the
ribosome one at a time.
– Each tRNA identifies the appropriate codon on the mRNA and add
this amino acid to the growing protein chain.
– The first tRNA is released and the ribosome moves one codon
length along the message, allowing the next tRNA to come into
place, carrying its amino acid. Energy in the form of ATP is required
at each step in the movement.
– As the ribosome moves along the mRNA , it eventually encounters
a ‘stop’ codon. At this point, the polypeptide chain is released.
Steps in protein synthesis…
Bacterial ribosomes have 3
sites that bind aminoacyltRNAs
A site (aminoacyl)
P site (peptidyl)
E site (exit) (largely on 50s)
Formation of the initiation
complex. (in bacteria)
Steps in protein synthesis…
First step in elongation
(in bacteria)
- binding of the second
aminoacyl-tRNA
Steps in protein synthesis…
Second step in elongation
(in bacteria): formation of
the first peptide bond
-peptide bond is formed
Steps in protein synthesis…
Third step in elongation (in
bacteria): translocatoin
-ribosome moves one codon
towards the 3’end of mRNA.
-the didpeptidyl-tRNA is now
entirely in the P site
-A site is open (for incoming
tRNA)
-uncharged tRNA moves first to
E site, then leaves.
Steps in protein synthesis
Termination of protein synthesis
in bacteria
-occurs in response to a termination
codon in A site.
-a Release Factor binds to A site,
leads to hydrolysis of ester linkage
between nascent polypeptide and
the tRNA.
-polypeptide thus released.
-mRNA, deacylated tRNA, and
Release Factor leave ribosome.
Ribosome dissociates into 30S and
50S subunits.
Most common forms of DNA damage:
• Bulges due to deletions or insertions
• Missing, altered, or incorrect base
• UV-induced pyrimidine dimers
• Strand breaks at phosphodiester bonds or within
deoxyribose rings
• Covalent cross-linking of strands
Mutations
• Mutation means alterations in DNA structure
that produce permanent changes in the genetic
information encoded therein.
• There is a strong link between accumulation
of mutations and the processes of aging and
carcinogenesis.
Recombinant DNA
• There are no cure for molecular diseases. But medical
scientists dream to correct these inborn errors by
replacing a nonfunctional gene on a human chromosome
with one that is functional. The research involves
experiments with recombinant DNA.
• Recombination consists of cleaving DNA chains,
inserting a new piece of DNA into the gap created by the
cleavage, and resealing the chains.
Gene mutation
Gene mutations are the changes in the base sequence of DNA
•
Substitution, addition, or deletion of one or more nucleotides in the DNA
molecule are called gene mutation
Example:
SHESAWTHEBADBOYHITTHEDOG
SHE SAW THE BAD BOY HIT THE DOG
SHE SAW THE BAD TBO YHI TTH EDO G—Addition of one letter
Sense
Nonsense
SHE SAW THE ADB OYH ITT HED O G —Deletion of one letter
Sense
Nonsense
SHE SAW THE BAE BOY HIT THE DOG—Replacement of one letter
Sense
Nonsense
Molecular diseases
Sickle cell anemia and other molecular diseases are the products
of mutation
Mutation may be harmful or beneficial: Sickle cell trait and sickle
cell anemia illustrate both the harm and benefit
- People with sickle cell trait have immunity to malaria, so in this
sense mutation is beneficial
- People with sickle cell anemia are very sick and often die
young, which certainly is a harmful effect of mutation
Xeroderma pigmentosum (XP)
• Disease caused by defect in the nucleotide-excision repair
system.
• People with XP are extremely light sensitive, and readily
develop sunlight-induced cancers.
• They also have neurological abnormalities, presumably due
to inability to repair certain lesions caused by high rate of
oxidative metabolism in neurons
DNA damage by radiation
• We are constantly exposed to radiation due to,
–near UV radiation (in sunlight)
–constant field of ionizing radiation in form of cosmic rays
–constant exposure to radiation from radioactive elements
(radium, plutonium, uranium, radon, 14C, and 3H)
–X-rays (medical and dental examinations)
–radiation therapy (cancer and other diseases)
UV and ionizing radiations cause about 10% of all DNA
damage caused by environmental agents
Oxidative damage to DNA…
• Oxidative damage is possibly the most
important source of mutagenic alterations in
DNA.
• Excited oxygen species such as hydrogen
peroxide, hydroxyl radicals, and superoxide
radicals arise during irradiation or as a byproduct
of aerobic metabolism
Oxidative damage to DNA
• Cells have elaborate defense system to destroy these
reactive species. Thses includes enzymes catalase and
superoxide dismutase that convert reactive oxygen
species to harmless products.
• A fraction of these species will escape cellular
defenses, and cause damage (ranging from oxidation of
deoxyribose and base moieties to strand breaks)
• Data related to this type of damage is not available.
However, every day DNA of each human cell exposed to
thousands of such reactions.
Hereditary nonpolyposis colon cancer (HNPCC)
• This type of cancer generally develops at
an early age.
• This is caused by defects in mismatch
repair. Defects in at least five different
mismatch repair genes can give rise to
HNPCC
Breast cancer
• Mostly occurs in women with no known
predisposition.
• At least 10% of cases are associated with defects
in two genes (Brca1 and Brca2) associated with
DNA repair.
• Women with defects in either of these genes have
a >80% chance of developing breast cancer.
DNA repair…
• Biological macromolecules are susceptible to
chemical alterations that arise from environmental
damage or errors during synthesis.
• For RNAs, proteins, or other cellular molecules,
most consequences of such damage are
circumvented through normal turnover (synthesis and
degradation).
DNA repair…
• However, integrity of DNA is vital to cell
survival and reproduction.
• Its information content must be protected over
the life span of the cell and preserved from
generation to generation.
DNA repair…
• DNA is the only molecule that, if
damaged, is repaired by the cell.
• Such repair is possible because the
information content of duplex DNA is
inherently redundant.
DNA repair…
• Human DNA replication has an error rate of
about three base-pair mistakes during copying
of 6 billion base pairs in the diploid human
genome.
- low error rate is due to DNA repair systems
that review and edit the newly replicated
DNA
• Firther, about 10,000 bases (mostly purines)
are lost per cell per day from spontaneous
breakdown in human DNA; the repair systems
must replace these bases to maintain the fidelity
of the encoded information.
DNA repair…
• Usually, the complementary structure of DNA
ensures that the information lost through damage to
one strand can be recovered from the other.
• However, even errors involving both strands can be
corrected through recombination.
• Double-stranded breaks (potentially the most
serious lesions) can be repaired by recombination
events.
Nucleic Acid Chemistry
• Double – helical DNA and RNA can be denatured
(extreme pH and heat)
• Nucleic acids from different species can form
hybrids
• Nucleotides and nucleic acids undergo non
enzymatic browning
• DNA is often methylated
• Long DNA sequences can be determined
Other functions of nucleotides
• Nucleotides carry chemical energy in cells.
• Nucleotides are components of many enzyme
cofactors.
• Some nucleotides are intermediates in cellular
communications.