Chapter 7a - DNA mutation and repair: Mutation and

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• Chapter 7a - DNA mutation and repair
• Mutation and adaptation
• Types of mutations
• DNA repair mechanisms

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• Mutation and adaptation
• Jean-Baptiste Lamarck (1744-1829)
• Proposed inheritance of acquired traits 1801.
• Induction by the environment also known as
  transformism, transmutation, or soft inheritance.
• Examples giraffes that acquire longer necks or
  athletes that build strong muscles pass these
  traits to their offspring.
• In genetic terms, Lamarckism is the idea that
  environmentally induced mutations could be passed
  to the offspring.

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• Mutation and adaptation
• Jean-Baptiste Lamarck (1744-1829)
• Ideas largely ignored or attacked during his
  lifetime.
• Never won the acceptance and esteem of his
  colleagues and died in poverty and obscurity.
• Today Lamarck is mostly associated with a
  discredited theory of heredity (Lamarckism
  persisted until 1930s/1940s).
• Interest in Lamarck has resurged with discoveries
  in the field of epigenetics.
• Epigenetics - heritable changes in gene
  expression or the phenotype caused by mechanisms
  other than changes in the underlying DNA sequence
  (? maternal effect).

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• Charles Darwin (1809-1882)
• Heritable adaptive variation results from random
  mutation and natural selection (1859, The Origin
  of Species).
• Contrary to Larmarck, inheritance of adaptive
  traits does not result from induction by
  environmental influences.
• But differential survival (selection) and
  heritable variation (arising from mutation in the
  DNA sequence).
• Years following Darwin and rediscovery of Mendel
  resulted in controversy (until 1930s/1940s) about
  the relative importance of mutation and
  selection.
• Largely resolved by theoretical and empirical
  work of Fisher, Haldane, and Wright (see chapter
  21 lectures).

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• Experimental test of Lamarcks inheritance of
  acquired traits
• Salvador Luria and Max Delbrück (1943)
• An E. coli population started from one cell
  should show different patterns of T1 resistance
  depending on which theory is correct.
• Lamarcks theory states that cells are induced to
  become resistant when T1 is added proportion of
  resistant cells should be the same for all
  cultures with the same genetic background.
• Mutation theory states that random events confer
  resistance to T1 duplicate cultures with the
  same genetic background should show different
  numbers of T1 resistant cells.

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Fig. 7.2, Fluctuating populations of E. coli
infected with T1 phage. Luria and Delbrück (1943)
Lamarck theory prediction proportions or
resistant cells are the same
Mutation theory prediction proportions are
function of genotypes
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• What is a mutation?
• Substitution, deletion, or insertion of a base
  pair.
• Chromosomal deletion, insertion, or
  rearrangement.
• Somatic mutations occur in somatic cells and only
  affect the individual in which the mutation
  arises.
• Germ-line mutations alter gametes and passed to
  the next generation.
• Mutations are quantified in two ways
• Mutation rate probability of a particular type
  of mutation per unit time (or generation).
• Mutation frequency number of times a particular
  mutation occurs in a population of cells or
  individuals.

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• Two types of point mutations
• Base pair substitutions.
• Transitions
• Convert a purine-pyrimidine to the other
  purine-pyrimidine.
• 4 types of transitions A ? G and T ? C
• Most transitions results in synonymous
  substitution because of the degeneracy of the
  genetic code.
• Transversions
• Convert a purine-pyrimidine to a
  pyrimidine-purine.
• 8 types of transversions A ? T, G ? C, A ? C,
  and G ? T
• Transversions are more likely to result in
  nonsynonomous substitution.

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Terminology describing mutations in protein
coding sequences Nonsynonymous/missense
mutation Base pair substitution results in
substitution of a different amino acid. Nonsense
mutation Base pair substitution results in a
stop codon (and shorter polypeptide). Neutral
nonsynonymous mutation Base pair substitution
results in substitution of an amino acid with
similar chemical properties (protein function is
not altered). Synonymous/silent mutation Base
pair substitution results in the same amino
acid. Frameshift mutations Deletions or
insertions (not divisible by 3) result in
translation of incorrect amino acids, stops
codons (shorter polypeptides), or read-through of
stop codons (longer polypeptides).
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Fig. 7.3, Types of base pair substitutions and
mutations.
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Fig. 7.3, Types of base pair substitutions and
mutations.
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Fig. 7.4, Effect of a nonsense mutation on
translation.
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• Reverse mutations and suppressor mutations
• Forward mutation
• Mutation changes wild type (ancestral) to mutant
  (derived).
• Reverse mutation (back mutation)
• Mutation changes mutant (derived) to wild type
  (ancestral).
• Reversion to the wild type amino acid restores
  function.
• Reversion to another amino acid partly or fully
  restores function.
• Suppressor mutation
• Occur at sites different from the original
  mutation and mask or compensate for the initial
  mutation without reversing it.
• Intragenic suppressors occur on the same codon

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• Intergenic suppressor genes
• Many function in mRNA translation.
• Each suppressor gene works on only one type of
  nonsense, missense, of frameshift mutation.
• Suppressor genes often encode tRNAs, which
  possess anti-codons that recognize stop codons
  and insert an amino acid.
• Three classes of tRNA nonsense suppressors, one
  for each stop codon (UAG, UAA, UGA).
• tRNA suppressor genes coexist with wild type
  tRNAS.
• tRNA suppressors compete with release factors,
  which are important for proper amino acid chain
  termination.
• Small number of read-through polypeptides are
  produced tandem stop codons (UAGUAG) are
  required to result in correct translation
  termination.

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Fig. 7.5, tRNA suppressor gene mechanism for
nonsense mutation.
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• Spontaneous mutations differ from induced
  mutations
• Spontaneous mutations can occur at any point of
  the cell cycle.
• Movement of transposons (mobile genetic elements)
  causes spontaneous mutations.
• Mutation rate 10-4 to 10-6 mutations/gene/gener
  ation
• Rates vary by lineage, and many spontaneous
  errors are repaired.

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Spontaneous mutations Different types DNA
replication errors Wobble-pairing T-G, C-A,
A-G, T-C Normal pairing typically occurs in the
next round of replication frequency of mutants
in F2 is 1/4. GT pairs are targets for
correction by proofreading and other repair
systems. Additions and deletions DNA loops out
on template strand, DNA polymerase skips bases,
and deletion occurs. DNA loops out on new
strand, DNA polymerase adds untemplated bases.
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Fig. 7.7, Mutation caused by mismatch wobble base
pairing.
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Fig. 7.8, Addition and deletion by DNA
looping-out.
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Spontaneous mutations Spontaneous chemical
changes Depurination Common A or G are removed
and replaced with a random base. Deamination Ami
no group is removed from a base (C ? U) if not
replaced U pairs with A in next round of
replication (CG ? TA). Prokaryote DNA contains
small amounts of 5MC deamination of 5MC produces
T (CG ? TA). Regions with high levels of 5MC are
mutation hot spots.
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Fig. 7.9, Deamination.
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Induced mutations Radiation (e.g., X-rays,
UV) Ionizing radiation breaks covalent bonds
including those in DNA and is the leading cause
of chromosome mutations. Ionizing radiation has
a cumulative effect and kills cells at high
doses. UV (254-260 nm) causes purines and
pyrimidines to form abnormal dimer bonds and
bulges in the DNA strands.
Fig. 19.10, Thymine dimers induced by UV light.
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• Induced mutations chemical mutagens
• Base analogs
• Similar to normal bases, incorporated into DNA
  during replication.
• Some cause mis-pairing (e.g., 5-bromouracil).
• Not all are mutagenic.

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Fig. 7.11a, Mutagenic efffects of 5-bromouracil
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Fig. 7.11b, Mutagenic efffects of 5-bromouracil
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• Induced mutations Chemical mutagens
• Base modifying agents, act at any stage of the
  cell cycle
• Deaminating agents
• Hydroxylating agents
• Alkylating agents

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Fig. 7.12, Base-modifying agents.
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Fig. 7.12, Base-modifying agents (cont.).
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• Induced mutations chemical mutagens
• Intercalating agents
• Thin, plate-like hydrophobic molecules insert
  themselves between adjacent base-pairs,
• Mutagenic intercalating agents cause insertions
  during DNA replication.
• Loss of intercalating agent can result in
  deletion.
• Examples proflavin, ethidium bromide

Fig. 7.13
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• Detecting environmental mutations Ames Test
  (after Bruce Ames)
• Ames Test is an inexpensive method used to screen
  possible carcinogens and mutagens.
• Histidine auxotroph Salmonella typhimurium
  (requires Histidine to grow) are mixed with rat
  liver enzymes and plated on media lacking
  histidine.
• Liver enzymes are required to detect mutagens
  that are converted to carcinogenic forms by the
  liver (e.g., procarcinogens).
• Test chemical is then added to medium.
• Control plates show only a small of revertants
  (bacteria cells growing without histidine).
• Plates innoculated with mutagens or
  procarcinogens show a larger of revertants.
• Auxotroph will not grow without Histidine unless
  a mutation has occurred.

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Fig. 7.14, Ames test.
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• Site-specific in vitro mutagenesis
• Method by which mutant alleles can be synthesized
  in the lab and transformed into cell culture and
  animals.
• Commonly used to study mutations of human genes
  in mice or other model organisms.
• One simple method relies on PCR
• Begin with 4 PCR primers 2 primers match the
  target sequence except where the mutation is
  desired, and 2 primers flank the region.
• Synthesize 2 PCR products in both directions from
  mutation site to cover full length of gene
• Remove primers, mix PCR products, and denature.
• Two PCR products now overlap self-anneal and
  extend full length products in a thermalcycler.
• Transform into cell or expression vector for
  further tests.

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Fig. 9.1, Site-specific mutagenesis using PCR.
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• DNA repair mechanisms
• Enzyme-based repair mechanisms prevent and repair
  mutations and damage to DNA in prokaryotes and
  eukaryotes.
• Types of mechanisms
• DNA polymerase proofreading - 3-5 exonuclease
  activity corrects errors during the process of
  replication.
• Photoreactivation (also called light repair) -
  photolyase enzyme is activated by UV light
  (320-370 nm) and splits abnormal base dimers
  apart.
• Demethylating DNA repair enzymes - repair DNAs
  damaged by alkylation.
• Nucleotide excision repair (NER) - Damaged
  regions of DNA unwind and are removed by
  specialized proteins new DNA is synthesized by
  DNA polymerase.
• Methyl-directed mismatch repair - removes
  mismatched base regions not corrected by DNA
  polymerase proofreading. Sites targeted for
  repair are indicated in E. coli by the addition
  of a methyl (CH3) group at a GATC sequence.

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Fig. 7.16 Nucleotide excision repair (NER) of
pyrimidine dimmer and other damage-induced
distortions of DNA
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Fig. 7.17 Mechanism of mismatch correction repair