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1 Prakash Pokhrel
Genetic materials and its organization into chromosome (DNA)
In this session...
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Identification of genetic material
Components of DNA
Structure of DNA
Replication
Damage and repair
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IntroductionThe progeny of organism develops characters similar to that
organism
The resemblance of offspring to their parents depends on the
precise transmission of principle component from one generation
to the next
That component is- The Genetic Material
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The genetic material of a cell or an organism refers to those materials found in the nucleus, mitochondria and cytoplasm, which play a fundamental role in determining the structure and nature of cell substances, and capable of self-propagating and variation.
What is genetic material?
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Four requirements for a genetic material
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• Must carry information– Cracking the genetic code
• Must self replicate– DNA replication
• Must allow for information to change
– Mutation
• Must govern the expression of the phenotype
– Gene function
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Identification of genetic material:
RNA
DNA
PROTEINDNA
The process of identification of genetic material began in
1928 with experiments of Griffith and concluded in 1952 with
the studies of Hershey and Chase.
Between these two experiments other three scientists, Avery,
Macloed and McCarty were did an experiment to identify the
genetic material.
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Discovery of Transformation in Bacteria:
In 1928, Frederick Griffith discovered bacterial transformation. He worked on Streptococcus pneumonieae (Pneumococcus) Pneumococci have various strains which can be classified by- 1. The presence or absence of a polysaccharide capsule2. The molecular composition of the capsuleWhen grown on blood agar medium, pneumococci with capsules are virulent and form large, smooth colonies and designated as typeIII S
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S pneumococci mutate to an avirulent form that has no capsules.
When grown on blood agar medium, these noncapsulated pnuemococci form small, rough-surfaced colonies and designated as typeII R
Based on the molecular composition of the capsule, these pneumococci cells are type I, II, III, and so forth.
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(Griffith,1928)
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Based on these observations he concluded that some of the cells of typeIIR had changed into typeIIIS due to influence of dead typeIIIS cells
He called this phenomenon as transformation
Principle Component of typeIIIS cells which induced the conversion of type IIR cells into type IIIS was named transforming principle
Griffith’s Conclusions:
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Griffith’s transforming principle was the genetic material
Transformation assay to identify actual biomolecule
Major constituents - DNA, RNA, proteins, carbohydrates & lipids
Made cell extracts from type IIIS cells containing each of these macromolecules
1944 - Avery, MacLeod & McCarty Identify the Genetic Material
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Avery, MacLeod, McCarty Experiment: The transforming principle is DNA
13(Avery, et al., 1944)
(Avery, et al., 1944) 14
The evidence presented supports the belief that a nucleic
acid of the deoxyribose type is the fundamental unit of the
transforming principle of Pneumococcus TypeIIIS.
(Avery, et al., 1944)
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What is DNA?
• nitrogen base and sugar make a nucleoside. • Phosphate group and a nucleoside make a
nucleotide.
•DNA is deoxyribo nucleic acid. A German chemist,Friedrich Miescher, discovered DNA in 1869.
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•DNA contains three main components (1) Phosphate (PO4) groups;
(2) Five-carbon sugars; and(3) Nitrogen-containing bases called purines and pyrimidines.
Components of DNA:
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Assembly into nucleotides
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Nucleotides linked in a chain
The phosphate group of one nucleotide is attached to the sugar of the next nucleotide in line.
• The result is a “backbone” of alternating phosphates and sugars, from which the bases project
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5’ PO4
PO4 5’
3’ OH
3’ OH
Structure of DNA:
• Two polynucleotide
chains are held
together by
hydrogen bonding
between bases in
opposing strands.
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Watson and Crick’s structure :
They proposed that DNA as
a right handed double helix
with two poly nucleotide
chains are coiled about one
another in a spiral.
(Watson and Crick,1953)21
The strands of DNA are antiparallel
The strands are complimentary
There are Hydrogen bond forces
There are base stacking interactions
There are 10 base pairs per turn
Properties of a DNA double helix
(Watson and Crick,1953)22
23 Watson and Crick with their model of DNA structure
Basis for double helix:
Rosalind Franklin’s DNA X-ray diffraction photograph.
Central cross mark indicates –helical structure of DNA.
Top and bottom dark bands indicates bases perpendicular to axis of molecule.
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Chargaff’s base pairing rule:
Percent of adenine = percent of thymine (A=T)
Percent of cytosine = percent of guanine (C=G)
A+G = T+C (or purines = pyrimidines)
(Chargaff et al.,1950)25
DNA Replication:
Replication is one of the most important requirement for a genetic material.
The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules.
It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’. (Watson and Crick,1953) 26
extreme accuracy of DNA replication is necessary in order
to preserve the integrity of the genome in successive
generations.
DNA has to be copied before a cell divides
DNA is copied during the S or synthesis phase of
interphase
New cells will need identical DNA strands
Biological significance:
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Models of DNA replication:
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Steps in DNA replication:
Initiation
Proteins bind to DNA and open up double helix
Prepare DNA for complementary base pairing
Elongation
Proteins connect the correct sequences of nucleotides
into a continuous new strand of DNA
Termination
Proteins release the replication complex
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Binding proteins prevent single strands from rewinding.
Helicase protein binds to DNA sequences called origins and unwinds DNA strands.
5’ 3’
5’
3’
Primase protein makes a short segment of RNA complementary to the DNA, a primer.
3’ 5’
5’ 3’
Proteins in replication:
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Overall directionof replication 5’ 3’
5’ 3’
5’
3’
3’ 5’
DNA polymerase III enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces
incorrect nucleotides.
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3’ 5’
3’ 5’
5’ 3’
5’ 3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
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5’
5’
3’ 3’ 5’
3’
5’ 3’
5’ 3’
3’
5’
Exonuclease activity of DNA polymerase I removes RNA primers.
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Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.
3’ 5’
3’
5’ 3’
5’ 3’
3’
5’
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Origin of replication:
Initiator proteins identify specific base sequences on DNA called sites of origin.
Prokaryotes – single origin site E.g E.coli Eukaryotes – multiple sites of origin (replicator)
Prokaryotes Eukaryotes
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Most eukaryotes except for budding yeast have ill-defined
origins of replication that rely on epigenetic mechanisms for
molecular recognition by initiator proteins.
Replication is initiated at multiple origins along the DNA
using a conserved mechanism that consists of four steps:
origin recognition, assembly of a prereplicative initiation
complex, followed by activation of the helicase and loading of
the replisome.
(Sclafani and Holzen,2007)36
Uni or bidirectionalReplication forks move in one or opposite directions
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Replication Fork
View of bidirectional movement of the DNA replication machinery
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Semi-discontinuous replication
Anti parallel strands replicated simultaneouslyLeading strand synthesis continuously in 5’– 3’Lagging strand synthesis in fragments in 5’-3’
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DNA synthesis only in 5’ 3’:
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Eukaryotic enzymes:
Five common DNA polymerases from mammals.
1. Polymerase (alpha): nuclear, DNA replication, no proofreading
2. Polymerase (beta): nuclear, DNA repair, no proofreading
3. Polymerase (gamma): mitochondria, DNA replication, proofreading
4. Polymerase (delta): nuclear, DNA replication, proofreading
5. Polymerase (epsilon): nuclear, DNA repair, proofreading
Polymerases vary by species.
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Model of DNA Replication:
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End-replication problem:
Every time a linear chromosome replicates, the laggaing strand at each end gets shorter by about 150 nucleotides. Because there is a minimum length of DNA needed for initiation of an Okazaki fragment.
DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication.
Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase binds to the terminal telomere repeat and catalyzes the addition of of new repeats.Compensates by lengthening the chromosome.
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DNA Damage and Repair:
DNA polymerase do great job during DNA replication by proof reading the new DNA strand.
But its not enough to maintain the 100% fidelity in replication.
Several kinds of damage occurs by endogenous and exogenous agents.
DNA has its own mechanisms to repair this damages and maintain the accuracy of copying mechanism.
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Natural polymerase errorEndogenous DNA damage
oxidative damage depurination
Exogenous DNA damageradiation
chemical adducts“Error-prone” DNA repair
Sources of damage
DNA Damage Response(DDR):
To respond to these threats, eukaryotes have evolved the
DNA Damage Response (DDR).
The DDR is a complex signal transduction pathway that has
the ability to sense DNA damage and transduce this
information to the cell to influence cellular responses to
DNA damage.
(Ciccia and Elledge, 2010)
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“Mutation is rare because of repair”
Over 200 human genes known to be involved in DNA repairMajor DNA repair pathways:1. Base excision repair (BER)2. DNA Mismatch repair (MMR)3. Nucleotide excision repair (NER)4. DNA strand break repair pathways:
Single strand break repair (SSBR)Double-strand break repair pathways (DSBR)Homologous Recombination (HR)Nonhomologous end joining (NHEJ)
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Direct reversal of damage - Photoreactivation (bacteria, yeast, some vertebrates - not humans) Two thymines connected together by UV light.
Excision Repair - removal of defective DNA. There are three distinct types1) base-excision2) nucleotide-excision3) mismatch repair
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Base-excision repair(BER):Presence of the Uracil in DNA is a great example of this typeSpecial enzymes replace just the defective base
snip out the defective basecut the DNA strandAdd fresh nucleotideLigate gap
N
N
NH2
O
O
H2
C
O
ON
HN
O
O
O
H2
C
O
O
deoxycytosine deoxyuracil
1’
2’3’
4’
5’
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5
6
CH3
thymine
glycosidic bond
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Nucleotide-excision repair(NER):
Same as previous except that-
It removes entire damaged nucleotide
Remove larger segments of DNA
Example:Xeroderma pigmentosum
• Extreme sensitivity to sunlight
• Predisposition to skin cancer
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Mismatch repair (MMR):Despite extraordinary fidelity of DNA synthesis, errors do
persistSuch errors can be detected and repaired by the post-
replication mismatch repair systemSpecial enzymes scan the DNA for bulky alterations in the
DNA double helixThese are normally caused by mismatched bases
A GA CC T
These are excised and the DNA repaired
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MMR also processes mispairs that result from heteroduplex DNA
formed during genetic recombination: act to exclude “homeologous”
recombination.
Repair involving two or more close sites in same heteroduplex occur
much more often on the same strand than the opposite strands.
Analysis of the pattern of repair suggest that repair tracks initiates at
mismatches and propagate preferentially in 5’ to 3’ direction.
(Wagner and Meselson, 1976)52
The problem of strand discrimination:
MMR can only aid replication fidelity if repair is targeted to newly synthesized strand
The cell has a mechanism of identifying new strand synthesis by leaving nicks that DNA. There are enzymes which scan these new regions looking for errors.
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Other forms of DNA damage:Depurination - the base is simply ripped out of the DNA molecule
leaving a gap.
Deamination - An amino group of Cytosine is removed and the base becomes Uracil.
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Basic mechanism is the same for all three types
1) Remove damaged region
2) Resynthesis DNA3) Ligate
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