This document summarizes DNA mutation and repair mechanisms. It discusses Lamarck and Darwin's theories of heredity and adaptation. It describes different types of mutations like substitutions, deletions, and frameshifts. Experiments by Luria and Delbrück tested Lamarck and Darwin's theories. The document also discusses DNA repair mechanisms in cells, spontaneous mutations from replication errors, and induced mutations from radiation, chemicals and intercalating agents. The Ames test is described to detect mutagens and carcinogens. Site-specific mutagenesis techniques like PCR can introduce mutations into genes.

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

2. Mutation and adaptation:
Jean-Baptiste Lamarck (1744-1829)
• Proposed “inheritance of acquired traits” ~1801; induction by the
environment; also known as transformism or transmutation.
• 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 associated with a discredited theory of heredity
(but Lamarckism persisted until 1930s/1940s).

3. 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
(originally arising from mutation).
• 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).

4. Experimental test of Lamarck’s “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.
1. Adaptive 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.
2. 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.

5. Fig. 7.2, Fluctuating populations of E. coli infected with T1 phage.
Luria and Delbrück (1943)
Adaptive theory prediction: proportions or resistant cells are the same
Mutation theory prediction: proportions are function of genotypes

6. 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:
1. Mutation rate = probability of a particular type of mutation per unit
time (or generation).
2. Mutation frequency = number of times a particular mutation occurs
in a population of cells or individuals.

7. Types of mutations:
Two types of point mutations:
1. Base pair substitutions.
1. 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.
2. Transversions
• Convert a purine-pyrimidine to a pyrimidine-purine.
• 8 types of transversions; A ↔ T, G ↔ C, A ↔ C, and G ↔ T
• Transversion more likely to result in nonsyn substitution.
2. Base pair deletions and insertions

8. Types of mutations in ORFs:
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).

9. Fig. 7.3, Types of base pair substitutions and mutations.

10. Fig. 7.3, Types of base pair substitutions and mutations.

11. Fig. 7.4, Effect of a nonsense mutation on translation.

12. Reverse mutations and suppressor mutations:
Forward mutation
Mutation changes wild type to mutant.
Reverse mutation (back mutation)
Mutation changes mutant to wild type.
• 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;
e.g., nearby addition restores a deletion
• Intergenic suppressors occur on a different gene.

13. 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.

14. Fig. 7.5, tRNA suppressor gene mechanism for nonsense mutation.

15. Spontaneous and induced mutations:
Spontaneous 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/generation
• Rates vary by lineage, and many spontaneous errors are repaired.

16. 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.

17. Fig. 7.7, Mutation caused by mismatch wobble base pairing.

18. Fig. 7.8, Addition and deletion by DNA looping-out.

19. Spontaneous mutations:
Spontaneous chemical changes
Depurination
Common; A or G are removed and replaced with a random base.
Deamination
Amino 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.

20. Fig. 7.9, Deamination.

21. Spontaneous and induced mutations:
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.

22. 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.

23. Fig. 7.11a, Mutagenic efffects
of 5-bromouracil

24. Fig. 7.11b, Mutagenic efffects
of 5-bromouracil

25. Induced mutations: Chemical mutagens
Base modifying agents, act at any stage of the cell cycle:
• Deaminating agents
• Hydroxylating agents
• Alkylating agents

26. Fig. 7.12, Base-modifying agents.

27. Fig. 7.12, Base-modifying agents (cont.).

28. 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

29. 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.

30. Fig. 7.14, Ames test.

31. 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 method relies on PCR:
1. Begin with 4 PCR primers; 2 primers match the target sequence
except where the mutation is desired, and 2 primers flank the
region.
2. Synthesize 2 PCR products in both directions from mutation site to
cover full length of gene
3. Remove primers, mix PCR products, and denature.
4. Two PCR products now overlap; self-anneal and extend full length
products in a thermalcycler.
5. Transform into cell or expression vector for further tests.

32. Fig. 9.1, Site-specific
mutagenesis using PCR.

33. 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 and 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.

34. Fig. 7.16 Nucleotide excision repair (NER) of pyrimidine dimmer
and other damage-induced distortions of DNA

35. Fig. 7.17 Mechanism of mismatch correction repair