DNA Damage and Repair

BIO 101
DNA DAMAGE AND REPAIR

© jcksanpascual 2020
Mutation
• Any heritable change in the DNA
• May have deleterious or (rarely) advantageous
consequences to an organism or its descendants
• Importance:
• Understanding of certain diseases and biological
phenomena
• Major source of genetic variation which fuels
evolutionary change
Types of Gene Mutations
• Point Mutation – substitution/addition/deletion in
the gene that may involve a single or multiple base
pairs
• Base Substitution
• Transition: Pu  Pu or Py  Py
• Transversion: Pu  Py
• Frameshift Mutation
• Base Addition: frameshift to the left
• Base Deletion: frameshift to the right
Effect of Point Mutations on
Amino Acid Sequence
• Silent mutation
• Mutated gene sequence results to the same
amino acid
• Neutral mutation
• Mutated gene sequence results in another amino
acid with similar chemical properties
Effect of Point Mutations on
Amino Acid Sequence
• Missense mutation
• Mutated gene sequence results in another amino
acid with a different chemical property
• Nonsense mutation
• Mutated gene sequence results into a stop codon
(or a stop codon changed into an amino acid-
coding codon)
MUTAGENESIS
Mutagenesis
• Spontaneous
• Occurs as a result of natural processes in cells
• Induced
• Occurs as a result of interaction of DNA with an
outside agent or mutagen
• Mutagen
• Chemical/physical agents that cause
mutation
Spontaneous Mutagenesis
1. Spontaneous replication errors
• Evades the proofreading function of the DNA
polymerase (mismatches)
2. Tautomerization
• Anomalous pairing between tautomeric bases
3. Replication Slippage
• In DNA with short repeated sequences
• Happens when a nucleotide from either the
template or daughter DNA loops out
• Results in frameshift mutation
3. Replication Slippage
4. Spontaneous
Depurination
• N-glycosidic bond
can be spontaneously
hydrolyzed at
physiological
temperature or
at pH 3
• Creates an apurinic
(aP) or baseless
site
5. Spontaneous Deamination
MUTAGENS
Induced Mutations
• Chemical mutagens
• Base analogues, base-modifying agents,
intercalating agents
• Physical mutagens
• UV radiation, ionizing radiation, heat
• Biological Agents of mutation
• transposable elements / transposons
• viruses and bacteria
Chemical Mutagens
1. Base analogues
• Structurally similar to the standard bases
• Can be incorporated into the nucleotides during
DNA replication
• Cause point mutations
• Eg.
• 5-bromouracil – an analogue of T
• 2-aminopurine – an analogue of A
Chemical Mutagens
• Mutagenic effect of 5-bromouracil
Chemical Mutagens
• Mutagenic effect of 5-bromouracil
Chemical Mutagens
• Mutagenic effect of
2-aminopurine
Chemical Mutagens
2. Base-modifying agents
• Deaminating agents
• HNO2 – deaminates A, C & G
• SO2 – converts C to U
• NaHSO3 – deaminates C
• Hydroxylating agents
• Hydroxylamine – hydroxylates C
Chemical Mutagens
2. Base-modifying agents
• Alkylating agents (adds Me or Et groups)
• Epoxides and tetraethyl lead
• Ethylmethane sulfonate (EMS) and
methylmethane sulfonate (MMS)
• Alkylate G
Chemical Mutagens
3. Intercalating agents
• flat molecules that insert themselves between
adjacent bases in the double helix, causing
distortion at the point of insertion.
Chemical Mutagens
Chemical Mutagens
Physical Mutagens
1. UV Radiation at 260 nm
• dimerization of adjacent pyrimidine bases
• cyclobutyl dimer / thymine dimer
• (6-4) lesion
• (6-4) photoproduct
• distorts helix as DNA bases are pulled closer

• extensive cleavage of H-bonds
• inhibits the advancing of the replication fork
Physical Mutagens
Physical Mutagens
• cytosine transformation to its imine tautomer –
pairs with A
• covalent joining of complementary strands due
to interchain dimerization
Physical Mutagens
2. Ionizing radiation
• X-rays, gamma rays, high speed e-s or alpha
particles; fast-moving neutrons
• More potent than UV
• Effects:
• formation of rare tautomeric enols
• removal of C from the DNA
• favored formation of the imine tautomer of C
• production of ss/ds breaks on the DNA
backbone
Physical Mutagens
3. Heat
• stimulates water-induced cleavage of the
β-N-glycosidic bond
• results in an AP / baseless site
• not normally mutagenic because cells have
effective systems for repairing nicks
Biological Agents of Mutations
1. Transposable Elements
• mobile pieces of DNA that can move from one
location in a genome to another
• presence of a transposon in a wild type gene
disrupts the normal function of that gene
2. Viruses
• can insert viral DNA into the genome and disrupt
genetic function
3. Bacteria (e.g. Helicobacter pylori)
• cause inflammation during which oxidative
species are produced that can damage DNA or
reduce efficiency of DNA repair systems
• H. pylori also produces nitroso-derivatives which
can modify bases
Mutator Genes
• Genes which, in their mutant state, increases
frequency of mutations in other genes
• Eg.
• DNA polymerase I and Dam methylase
• DNA repair enzymes
• Keep mutation frequency low
• When their genes are mutated,
• No functional repair enzyme is produced
• mutation frequency in various genes
increases
DNA REPAIR MECHANISMS
DNA Repair Mechanisms
• Direct Repair
• Excision Repair
• Mismatch Repair
• Double Strand Break Repair
• SOS Response
Direct Repair
• direct reversal
• acts directly on damaged nucleotides, converting
each one back to its original structure
• does not require a template
• three types of damage that are directly repaired:
• nicks
• alkylation damages
• cyclobutyl dimers
Direct Repair: Nicks
• Nicks
• Broken
phosphodiester bonds
• result from exposure to
ionizing radiation
• can be directly
repaired by DNA ligase
Direct Repair: Alkylation Damage
• repaired through enzymatic transfer of alkyl group
from the nucleotide to the repair enzyme’s own
polypeptide chain
• e.g.
• Ada enzyme of E. coli
• human MGMT
(O6-methylguanine-DNA
methyltransferase)
• Suicide enzyme
Direct Repair: Cyclobutyl Dimers
• repaired by DNA photolyase in many bacteria and a
few eukaryotes
• Involves cleavage of the cyclobutyl ring to regenerate
the adjacent thymine residues
Direct Repair
Base Excision Repair
• Excision/removal of a single damaged base and then
repair through re-synthesis
• Enzymes needed:
• DNA glycosylase
• breaks β-N-glycosidic bond between a
damaged base and sugar component
• creates AP site
• AP endonuclease
• cuts phosphodiester bond on the 5’ of AP site
• DNA polymerase β and DNA ligase
Base Excision Repair
Nucleotide Excision Repair
• repairs damage affecting longer strands (2-30 bases)
• used by the cell for bulky DNA damage
• NER in bacteria
• mediated by gene products of uvrA, uvrB and
uvrC
• NER in eukaryotes
• involves XPA,XPB,XPC….XPG proteins
• CSA and CSB proteins
• ERCC 1, RPA and Rad23 proteins
Nucleotide Excision Repair
Mismatch Repair
• Corrects a wrong nucleotide incorporated during
replication (mismatched nucleotide)
• Methyl-directed repair process
• Takes advantage of methyl groups at specific sites
of the DNA strand
• essential MMR proteins
• Prokaryotes Mut S, Mut H, Mut L
• Eukaryotes Msh2/Msh6 ; Msh2/Msh3
Mismatch Repair in E. coli
SOS Response
• DNA repair system in bacteria
• An inducible repair system
• activated when bacteria is treated with DNA
damaging agents that arrest DNA replication and
cell division
• Allows DNA replication to proceed through a highly
damaged region
SOS Response: Initiation
1. activation of RecA protein
• Binds to ssDNA that can’t be replicated due to
damage
• acts as a protease
2. degradation and inactivation of LexA by RecA
• LexA is the repressor of various genes needed for
DNA repair via SOS response
3. expression of uvrA and umuCD genes
• uvrA gene – enzymes for NER
• umuCD gene – DNA polymerase V
DNA Polymerase V
• Catalyzes elongation of
daughter DNA strand
despite damages in the
template strand
• Able to use damage
terminus as primer for
further strand elongation
• Exhibits higher chances
of incorporating wrong
nucleotides – error prone
DNA replication.

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BIO 101

• distorts helix as DNA bases are pulled closer

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