Genetics- related to DNA as the genetic material, its damage followed by the repair mechanism

1. DNA As The Genetic Material,
DNA Damage & Repair Mechanisms.
台大農藝系 遺傳學 601 20000
Preety Sweta Hembrom.
M.Sc. Genomic science.
Central University Of Kerala
Chapter 2
slide 1
GENETICS
TOPIC:

2.  The genetic material must perform three
essential function:
1. The Genotypic function,
2. The Phenotypic function,
3. The Evolutionary function.
 This means that genetic material must
replicate in order to transmit copies from
parents to offspring.
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3.  In the early 1900s, chromosomes were shown
to be the carriers of hereditary information.
 In eukaryotes they are composed of both
DNA and protein, and most scientists initially
believed that protein must be the genetic
material.
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4.  Chromosome consists of protein and nucleic
acid
 Protein v.s. nucleic acid
Protein: 20 kinds of amino acid
Nucleic acid: 2 types – DNA & RNA
 During the 1940s and early 1940s through the
result of elegant experiments it was clearly
established that genetic information is stored
in nucleic acids & not in proteins.
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5.  Frederick Griffith in 1928 performed
experiment with Streptococcus pneumoniae
bacteria in mice.
 This showed that something passed from
dead bacteria into nearby living ones,
allowing them to change their cell surface.
 He called this agent the transforming
principle, but did not know what it was or
how it worked.
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7.  In 1944, Avery, MacLeod and McCarty published results
of a study that identified the transforming principle
from S. pneumoniae.
 Chemically separate the components (e.g., protein,
nucleic acids) and determine which was capable of
transforming live S. pneumoniae cells.
 Only the nucleic acid fraction was capable of
transforming the bacteria.
 nucleic acid fraction was contaminated with proteins.
 The researchers treated this fraction with either RNase
or protease and still found transforming activity, but
when it was treated with DNase, no transformation
occurred, indicating that the transforming principle was
DNA.
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9.  1. More evidence for DNA as the genetic material came
in 1953 with Alfred Hershey and Martha Chase’s work on
E. coli infected with bacteriophage T2.
 2. In one part of the experiment, T2 proteins were
labeled with 35S, and in the other part, T2 DNA was
labeled with 32P.
 3. The 35S-labeled protein was found outside the
infected cells, while the 32P-labeled DNA was inside the
E. coli, indicating that DNA carried the information
needed for viral infection. This provided additional
support for the idea that genetic inheritance occurs via
DNA.
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10. 10

11. A BRIEF INTRODUCTION:
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12.  Nucleic acids are composed of nucleotides.
 Each nucleotide consists of:
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1. A Phosphate group,
2. 5 carbon sugar &
3. A cyclic nitrogen containing base.
In DNA:
 Sugar is 2-deoxyribose
 Four bases are adenine, guanine, thymine &
cytosine.

13. 13

14.  The faithful transmission of genetic material
from one cell to another is based on the
ability to replicate.
 The process of DNA replication is based on
the complementary nature of the strands
that makeup DNA duplex.
 These strands are held together by hydrogen
bonds between specific base pairs- A paired
with T & G paired with C.
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15.  Thus the expression of genetic information is
that the flow of information is from DNA to
RNA to polypeptide which is known as:-
Central Dogma Of Biology
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16. 16

17.  DNA is easily damaged under normal
physiological conditions.
 The return of damaged DNA to its normal
sequence and structure is called Repair.
 Many different kinds of physical & chemical
agents damage DNA. Some of these are:-
1. Endogenous agents
2. Exogenous agents
 Cells usually can survive DNA damage
provided that the replication and
transcription machinery can still perform
their functions.
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18. Hence, agents that damage DNA can be
mutagenic, cytotoxic or both.
DNA damaging agents that cause mutations
are called Mutagens.
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19.  Mutations are inherited changes in the
genetic material which provide new genetic
variation that allows organisms to evolve.
 Mutations occur in 2 ways:
1. Spontaneous mutations
2. Induced mutation
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20.  Damages are physical abnormalities in the
DNA, such as single and double strand breaks
etc.
In DNA damage transcription of a gene
can be prevented & thus translation as well
as replication of a gene will also be blocked
& the cell may die.
 Mutation is a change in the base sequence of
the DNA. It is replicated when the cell
replicate.
 Cause alteration in protein function &
regulation.
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21.  Although distinctly different from each
other, DNA damages & mutations are related
to each other because DNA damage often
cause errors of DNA synthesis during
replication or repair, these errors are a
major source of mutation.
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22.  The 4 major types of DNA damages are:
1. Radiation damage
2. DNA instability in water
3. Oxidative damage
4. Alkylation damage
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23.  The high energy electromagnetic radiation to
the exposure of which cell experience
considerable damage to their DNA are:
1. Ultraviolet light:
 The major type of damage caused by UV light
is divided into three bands:
I. UV-A (321-400 nm)
II.UV-B (296-320 nm)
III.UV-C (100-295 nm)
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24.  Two major photoproducts account for nearly
all of the UV induced DNA damage are:
a) Cyclobutane pyrimidine dimer- 75% of the UV
induced damage. Formed by introducing 2
new bonds between adjacent pyrimidines (C
& T) on the same DNA strand.
b) (6-4) photoproducts- Remaining of the UV
induced damage. Formed by introducing a
bond between the c-6 atom of one
pyrimidine & the c-4 atom of atoms of
adjacent pyrimidine on the same DNA strand.
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25. 2. X- Rays
3. Gamma Rays
 Through these direct damage takes place
when DNA or water tightly bound to it
absorbs the radiation.
Indirect damage takes place when
water or other molecules surrounding the
DNA absorbs the radiation & form reactive
species that then damage DNA.
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26.  DNA is damaged by hydrolytic cleavage
reactions.
 DNA has 2 kinds of bonds with the potential
for hydrolytic cleavage:
1. Phosphodiester bonds- Introduces a nick into
a DNA strand.
2. N- glycosyl bond- leads to the formation of
an abasic site also known as AP site. AP site
sensitizes the neighbouring 3’-
phosphodiester bond to cleavage.
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27.  Two of the oxidized base products that
cause damage are:
1. 8- oxoguanine (oxoG)- Base pair with A or
C. If uncorrected 8- oxoG- A base pair will
be replicated to form A-T base pair causing
transversion mutation.
Guanine oxidizes to 8-oxoguanine,
causes single & double strand breaks.
2. Thymine glycol- Inhibits DNA replication &
is therefore cytotoxic.
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28.  Alkylating agents damage DNA by
transferring alkyl groups to centers of
negative charge.
 Formation of monoadduct.
 Some of the DNA methylating agents are:
1. Dimethylguanosine
2. Dimethylsulfate.
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29.  There are five types of DNA repair
mechanisms:
1. Light- Dependent repair.
2. Excision repair.
3. Mismatch repair.
4. Post replication repair.
5. SOS response.
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30.  Carried out by light activated enzyme called
photolyase.
 Production of thymine dimers.
 DNA photolyase recognises & binds to
thymine dimers in DNA & uses light energy to
cleave the covalent cross- links.
 Photolyase also splits cytosine dimers &
cytosine- thymine dimers.
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32.  Two major types of excision repair:
I. Base-Excision repair- Remove abnormal or
modified bases from DNA.
II. Nucleotide-Excision Repair- Remove larger
defects like thymine dimers.
Base- Excision Repair:-
 Initiated by a group of enzymes- DNA
glycosylases( recognize abnormal bases in
DNA).
 The glycosylases cleave glycosidic bond
b/w the abnormal base & 2-deoxyribose
creating AP sites.
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33.  AP sites are recognized by enzymes- AP
endonucleases
 DNA polymerase then replaces the missing
nucleotide according to the specifications of
the complementary strand.
 Dna ligase seals the nick.
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Base Excision Repair (BER)
Variety of DNA glycosylases,
for different types of
damaged bases.
AP endonuclease recognizes
sites with a missing base;
cleaves sugar-phosphate
backbone.
Deoxyribose
phosphodiesterase removes
the sugar-phosphate lacking
the base.

35. Nucleotide Excision Repair:-
 Removes bulky DNA lesions that distort the
double helix.
 An enzyme complex recognizes the distortion
resulting from damage.
 Additional enzymes separate the two nucleotide
strands at the damaged region, & single strand
binding proteins stabilize the separated strands.
 The sugar phosphate backbone is cleaved on both
sides of the damage.
 Part of the damaged is peeled away & the gap is
filled by DNA polymerase & sealed by DNA ligase.
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36. Chapter 2
slide 36
Nucleotide Excision Repair
Excinuclease cuts on either
side of damage (~20 nt
altogether).
Strands unwound by
helicase.

37.  Many incorrectly inserted nucleotides
detected by proofreading are corrected by
mismatch repair.
 Enzymes cut out the distorted section of the
newly synthesized strand of DNA & replace
it with new nucleotides.
 The proteins that carry out this in E.coli
differentiate b/w old & new strands of DNA
by the presence of methyl group.
 Adenine nucleotides in GATC sequence is
methylated.
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38.  The mismatch repair complex brings the
mismatch bases close to the methylated
GATC sequence & the new strand is
identified.
 Exonucleases remove nucleotides on the new
strand b/w the GATC sequence & the
mismatch.
 DNA polymerase then replaces the
nucleotides correcting the mismatch & DNA
ligase seals the nick in the sugar phosphate
backbone.
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40.  Takes place after replication.
 When DNA polymerase III encounters a
thymine dimer in a template strand, its
progress is blocked.
 The damaged DNA is repaired by a
recombination- dependent repair process
mediated by E.coli recA gene product.
 The recA protein binds to the single strand of
DNA at the gap & mediates pairing with the
homologous segment of the sister double
helix.
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41.  The gap opposite the dimer is filled with the
homologous DNA strand from sister DNA
molecule.
 The resulting gap in the sister double helix is
filled by DNA polymerase
 And the nick is sealed by DNA ligase.
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42.  Discovered by Miroslav Radman in 1975.
 Two regulatory protein that controls SOS
response- LexA & RecA protein.
 Synthesized at low background levels.
 LexA binds to the DNA region that regulate
the transcription of the genes that are
induced during the SOS response.
 When the cells are exposed to UV lights or
other agents that cause DNA damage, the
RecA protein binds to the ss region of DNA.
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43.  The interaction of RecA with DNA activates
RecA, which then stimulates LexA to
inactivate itself by self-cleavage.
 With LexA inactivate, the level of expression
of the SOS genes increases & the error prone
repair system is activated.
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