Mutation

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Mutation

• A mutation is a change in the normal base pair
  sequence

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Commonly used to define DNA sequence changes that
alter protein function
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Mutation

• DNA replication is extremely accurate, BUT
• Errors in polymerization occur ??????? ?????
  ?????????
• Environmental factors, such as chemicals and
  ultraviolet radiation, can alter DNA ???????
  ?????? ??????? ????????
• Irreversible changes to the cellular DNA can be
  lethal
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• Non-lethal changes can cause a heritable
  alteration of the genetic information, called a
  mutation
• Genetic changes are more noticeable in germ line
  cells than in somatic cells
• Systems exist for the repair of damaged DNA

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A human DNA repair defect Xeroderma pigmentosum
Multiple skin cancers due to unrepaired UV damage
to DNA
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A human DNA repair defect Bloom's syndrome
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Xeroderma pigmentosum
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Xeroderma Pigmentosum (XP) and DNA Repair Defects

• XP is an autosomal recessive disease associated
  with dry skin, freckling, corneal ulceration, and
  skin tumors
• Many patients die before age 30 from metastases
  of malignant skin tumors
• One form of XP is produced by a defect in the
  human endonuclease that removes pyrimidine dimers
• Mutations in at least seven other genes involved
  in repairing UV-damaged DNA can cause XP

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Spontaneous mutagenesis and errors in DNA
replication

• DNA replication must accurately replicate 6 x 109
  base pairs every time a human cell divides
• ??? ????? ?? ?? ?????? ???? ?????? ??????????

• In man we see one new mutation per gene per
  100,000 cells per cell cycle. (spontaneous
  mutation frequency)
• ???? ?????? ??? ??? ??? ??? ?????? ??.

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DNA polymerases have two main methods for
ensuring accuracy
(a) Base selection
Only AT and GC base pairs fit properly in the
active site of the polymerase
(b) Proofreading
If a wrong base is inserted, then it is removed
and replaced with the correct one before the next
one is added
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Induced mutagenesis
Can be caused by environmental agents that damage
DNA

• UV light
• X-rays and g-rays
• Chemical carcinogens e.g. cigarette smoke

DNA damage can lead to mutations unless it is
removed by DNA repair enzymes Unrepaired damage
can have serious consequences
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1000
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Somatic vs. germ line mutations

• Somatic ((??? mutations can lead to cancer
• Germ (???/??????) line mutations can lead to
  birth defects (??? ????)
• Most mutations cause neither
• Some fall in non-coding DNA
• Others are silent

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(?????-??? ?? ?????????)
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????? ?????
(UV)
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• 1. ??, ?????? ????? ?? ?????? ????.
• 2. ??, ??? ?????? ?????? ?????? ????, ???? ?????
  ???? ?????? ?? ?????.
• 3. ????? 1 2 ??????.
• 4. ??? ????? ?????????? ?? ??? ????? ????? ?????.

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Natural causes of mutations??????? ??????? ?"?
???? ????

• Base tautomerization ??????? ?????????
• UV damage ???? ?????
• Spontaneous deamination - ?????????

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????????? - ?????? ?????????
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?? ????
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define the following types of mutations

• Transitions
• Transversions
• Multisite mutations including
• Inversions
• Duplications
• Deletions
• Insertions
• Substitutions

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define the following types of mutations

• Point mutations including
• Substitutions
• Insertions
• Deletions
• Duplications
• Inversions

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define the following types of mutations

• Point mutations including
• Substitutions
• Insertions
• Deletions
• Duplications
• Inversions

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define the following types of mutations

• For substitutions you should be able to further
  define
• Missense mutations
• Nonsense mutations
• Samesense mutations

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Point mutations

• Involve only one or a few nucleotides
• Arise during DNA replication
• Require two errors
• An error during DNA replication
• Failure to correct that error

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Types of point mutations

• Substitutions GATC CATC
• Insertion GATC GGATC
• Deletion GATC GTC
• Duplication GATC GAGATC
• Inversion GATC GTAC

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Types of substitutions

• Missense Results in an amino acid substitution
• Nonsense Results in a stop codon (TAG, TAA,
  TGA)
• Samesense No effect (silent mutation)

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What is the first defense against mutations?

• 3 to 5 exonuclease activity of the polymerases

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UV Radiation and Pyrimidine Dimers

• UV radiation (200-300 nm) induces the formation
  of dimers between adjacent thymine residues
• Cytosine-cytosine and thymine-cytosine dimers
  occur less frequently
• These pyrimidine dimers disrupt the structure of
  the double helix, blocking replication until the
  lesion is repaired

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???????? ??????? ?DNA ????? ????? ????????????
??? ?????? ??????? ?????? ?? ??????
???????? ???????? ?? ??????? ?????? ???????
??????? ?????? ??????? ?????? ?? ??????
?????? ??????? ???????? ?DNA ????? ?????????
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Mutations and Cancer

• Mutations that affect nonessential DNA or that
  have negligible effects on a genes function are
  called silent mutations
• Many mutations are detrimental, and a correlation
  exists between the accumulation of mutations and
  cancer
• New chemicals, such as pharmaceuticals, must be
  tested for carcinogenic potential
• Standard animal tests for carcinogenesis are
  lengthy and expensive
• The simple Ames test measures the potential of a
  given chemical to promote mutations in a
  specialized bacterial strain

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The Ames Test for Mutagenesis

• S. typhimurium having a defect in histidine
  biosynthesis are plated on a histidine-free
  medium
• Chemical to be tested is placed on a disk of
  filter paper in the center of the culture plate
• Revertant bacterial colonies are caused by
  mutagens
• 80 to 90 of compounds found to be carcinogenic
  in animal tests are mutagens in the Ames test

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Intercalating Agents

• Intercalating agents slip in between stacked base
  pairs
• Distance between base pairs is doubled by an
  intercalating agent
• Insertions or deletions of one or more
  nucleotides can occur during the replication of
  such distorted DNA

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Chemical Mutagens

• Classes of damage produced by chemical mutagens
• Point mutations one base pair is replaced by
  another
• Insertions or deletions one or more nucleotide
  pairs are inserted in or deleted from DNA
• Important types of chemical mutagens
• Alkylating agents ?????????
• Deaminating agents ??????????
• Intercalating agents ???????????

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???????? - Deamination of Cytosine
About 3 of the cytosine residues are methylated
(intentionally).

• Nitrous acid can be formed from nitrite and
  nitrate salts
• Nitrous acid oxidatively deaminates aromatic
  amines
• Cytosine is converted to uracil upon treatment
  with nitrous acid, causing a GC to AT transition
• Cytosine sometimes spontanteously deaminates to
  uracil, indicating why DNA contains thymine
  rather than uracil

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???? ???? ??? ???? ?????? ??????? "??????" ?"? BER
About 50 of the time the G is corrected to A
resulting in a mutation
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Alkylating Agents
?????????

• Alkylating agents such as dimethyl sulfate are
  electrophiles
• Such agents react most often with the
  nucleophilic N7 position of the guanine base
• Alkylation at N7 can generate an apurinic site in
  DNA
• Point mutations occur when guanine is replaced by
  another base
• DNA methylation can be useful

?????? ?"? BER
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Chemical mutagens????? ???????? ???????
????????

• Chemicals that accelerate the deamidation
  reaction ?????? ?????????
• Base analogues ??????? ???????
• Alkylating agents ?????? ????????
• Intercalation agents - ????????????

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Incorrect bases can swing out facilitating
identification by the DNA repair apparatus
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???? ???? ??? ???? ?????? ??????? "??????" ?"? BER
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Alkylating Agents
?????????

• Alkylating agents such as dimethyl sulfate are
  electrophiles
• Such agents react most often with the
  nucleophilic N7 position of the guanine base
• Alkylation at N7 can generate an apurinic site in
  DNA
• Point mutations occur when guanine is replaced by
  another base
• DNA methylation can be useful

?????? ?"? BER
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Intercalating Agents

• Intercalating agents slip in between stacked base
  pairs
• Distance between base pairs is doubled by an
  intercalating agent
• Insertions or deletions of one or more
  nucleotides can occur during the replication of
  such distorted DNA

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Overview of DNA repair pathways

• A. Base excision repair
• B. Nucleotide excision repair.
• C. Mismatch repair
• D. Double-strand break repair by
• homologous recombination.
• E. Double-strand break by end joining

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?"? DNA ????????
???? ??? ????? ????? ????????? ?????? ??? ??????
?"? MR
??????? ????? ?????? ????????? ?????? ??? ??? ?"?
BER ?? NER
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• Three general types of DNA repair pathways
• Direct Repair
• Base excision Repair
• Nucleotide excision repair

Direct repair
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DNA Repair

• Many DNA repair mechanisms exist, even in E. coli
• Systems include simple mechanisms to reverse base
  modifications and multienzyme systems
• O6-methylguanine-DNA methyltransferase transfers
  the methyl group from O6-alkylated guanine to one
  of its Cys residues
• DNA photolyases reverse pyrimidine dimers
• Found in both prokaryotes and eukaryotes, but not
  humans
• Contain cofactors that use light energy to
  mediate electron transfer to the dimer, reversing
  the cyclization

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Base excision repair
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uvrAB recognize the unpair bases
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NER
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Base-Excision Repair

• DNA glycosylases cleave the glycosidic bonds of
  altered nucleotides, leaving an apurinic or an
  apyrimidinic (AP) site
• Such sites also commonly occur through
  spontaneous depurination
• Deoxyribose residue is cleaved by an AP
  endonuclease
• DNA polymerase removes several residues and fills
  in the gap
• Nick sealed by DNA ligase

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Repair of the daughter strand
Nickase and exonuclease I
G
GATC
Parent
CTAG
New
DNA Pol I and ligase
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Base-excision repair?????

• Deaminated bases are recognized
• Enzymes remove the base

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Mismatch repair
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Occurs just after replication Improves accuracy
102 - 103 fold Must distinguish the parent from
the daughter strand
Mismatch repair ???? ??? ??? ?????
?????????. ???? ?????? ??? ???? ??? ???? (?"?
???????). ???? ????? ?????? ????? ???? ????
???????. ???? ???? ???????? ?? 100-1000.
Occurs just after replication Improves accuracy
102 - 103 fold Must distinguish the parent from
the daughter strand
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?? ????? ??? ????? ??? ??????? ?-mismatch repair
(MR) ???? ?????? ?-nucleotide excision repair
(NER) ?

• 1. ????? ?????? ??? ???? ????? ???
• 2. ?-MR ???? ???? ?-G1 ???? ??-NER ???? ?-M
  ?????? ???.
• 3. ?-MR ???? ???? ?-M ???? ??-NER ???? ?-G1
  ?????? ???.
• 4. ?-MR ???? ???? ?-G0 ???? ??-NER ???? ?-G1
  ?????? ???.

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Instability Mutation and DNA repair

• DNA repair
• E-coli mismatch repair
• Human mismatch repair

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Instability Mutation and DNA repair

• DNA repair
• Human mismatch repair

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Mismatch repair

• Occurs just after replication
• Improves accuracy 102 - 103 fold
• Must distinguish the parent from the daughter
  strand

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What happens when mismatch repair fails in humans?

• Missing enzymes homologous to MutS and MutL
• Patients usually die by age 30
• Disease hereditary nonpolyposis colorectal (???
  ??) cancer (HPCC)
• 1 in 200 people affected

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Nucleotide Excision Repair (NER)

• NER involves the removal of an oligonucleotide
  containing a lesion and replacement of the
  resulting gap
• Three enzymatic activities are responsible for
  this process in E. coli
• UvrABC endonuclease
• DNA Pol I
• DNA ligase
• NER mechanisms are similar in humans, but are
  much more complex

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Nucleotide excision repair
????? ?????? ?? ????? ?? ????? ????????
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?? ??????? ???? ????? ?? ?????? ?- 1NER
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?? ??????? ???? ????? ?? ?????? ?- 2 NER
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3 NER
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(NER) Nucleotid Excision Repair
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Nucleotide-excision repair?????

• Enzymes recognize a kink in the DNA
• Nicking
• Removal of the damaged strand
• DNA polymerization
• Ligation

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Excision repair of thymine dimers (UV-induced)
Repair enzymes remove damaged region
A C G
A T A A C T G C
Gap refilled by DNA polymerase and DNA ligase
Correct sequence restored
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Double strand breaks can give rise to
translocations
Novel genes formed at the fusion junctions can
have deleterious effects
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Homologous End-Joining recovers information from
the homologous chromosome
Information lost
A
Crossing over
Information restored
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Homologous End-Joining recovers information from
the homologous chromosome
A
Note The sequence of the paternal chromosome is
now identical to the maternal chromosome in the
repaired region -- ie. heterozygosity is lost
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Occurs During Meiosis
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Meiotic recombination results in swapping of
large regions between homologous chromosomes
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Overview of DNA repair pathways

• A. Base excision repair
• B. Nucleotide excision repair.
• C. Mismatch repair
• D. Double-strand break repair by
• homologous recombination.
• E. Double-strand break by end joining

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???? ?????? ?? ????? RecA
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Post replication recombination repair
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Post replication recombination repair
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Double-strand break repair
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Post replication recombination repair
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????? ?????? ?? ????? ????? ????????
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SOS repair
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Overview of DNA repair pathways

• A. Base excision repair
• B. Nucleotide excision repair.
• C. Mismatch repair
• D. Double-strand break repair by
• homologous recombination.
• E. Double-strand break by end joining

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Homologues recombination
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