dna : a genetic material, replication damage and repair

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Anilkumar C

PALM 3001

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DNA: A genetic material, its

replication, damage and repair

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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Introduction

The 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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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 capsule

2. The molecular composition of the capsule

When 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 geneticmaterial

Transformation assay to identify actual biomolecule

Major constituents - DNA, RNA, proteins,carbohydrates & lipids

Made cell extracts from type IIIS cells containingeach of these macromolecules

1944 - Avery, MacLeod & McCarty Identify the

Genetic Material

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Avery, MacLeod, McCarty Experiment:

The transforming principle is DNA

14(Avery, et al., 1944)

(Avery, et al., 1944) 15

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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Genetic information is transmitted by DNA only

The final evidence that DNA transmits genetic

information was provided by Hershey and Chase in

1952.

They experimented with T2 bacteriophages, viruses

that attack bacteria.

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(Hershey and Chase, 1952)

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(Hershey and Chase, 1952)

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• The sulphur containing protein of resting phage particles is

confined to a protective coat that is responsible for the adsorption

to bacteria, and functions as an instrument for injection of the

phage DNA into the cell. This protein probably has no role in

growth of intracellular phage. The DNA has some function.

Their conclusion:

(Hershey and Chase, 1952)

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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)26

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)

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28 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)

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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)

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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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Semiconservative model of DNA replication

(Meselson and Stahl,1958)34

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 direction

of replication5’3’

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 - oriC

Eukaryotes – multiple sites of origin (replicator) E.g.

yeast(ARS)

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)

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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 simultaneously

Leading strand synthesis continuously in 5’– 3’

Lagging strand synthesis in fragments in 5’-3’

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DNA Replication Fork

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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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Replication of circular DNA in E. coli:

1. Two replication forks

result in a theta-like

() structure.

2. As strands separate,

positive supercoils

form elsewhere in the

molecule.

3. Topoisomerases

relieve tensions in the

supercoils, allowing

the DNA to continue

to separate.50

1. Common in several bacteriophages

including .

2. Begins with a nick at the origin of

replication.

3. 5’ end of the molecule is displaced and

acts as primer for DNA synthesis.

4. Can result in a DNA molecule many

multiples of the genome length

5. During viral assembly the DNA is cut

into individual viral chromosomes.51

Rolling circle model of DNA Replication:

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 repair

Major 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 types

1) base-excision

2) nucleotide-excision

3) mismatch repair

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Base-excision repair(BER):

Presence of the Uracil in DNA is a great example of this type

Special enzymes replace just the defective base

snip out the defective base

cut the DNA strand

Add fresh nucleotide

Ligate gap

N

N

NH2

O

O

H2

C

O

ON

H

N

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 dmaged 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

persist

Such errors can be detected and repaired by the post-

replication mismatch repair system

Special enzymes scan the DNA for bulky alterations in the

DNA double helix

These are normally caused by mismatched bases

A G

A C

C 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)

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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 DNA

3) Ligate

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