DNA mutation and repair mechanisms in E. Coli include: 1) Spontaneous DNA damage such as base pair changes from rare tautomeric forms and base deamination. 2) Physical and chemical mutagens that damage DNA bases and cause breaks through oxidation, radiation, alkylation, and intercalation. 3) Four main DNA repair pathways - direct reversal, excision repair, recombinational repair, and the SOS response which is error-prone. 4) Mutations are point mutations like transitions, transversions, or frameshifts that change single DNA bases and cause protein alterations through modified codons.

1. DNA MUTATION AND
REPAIR

2. Machanism Cumulative error frequency
Base pairing ~10-1 - 10-2
DNA polymerase actions (including base ~10-5 - 10-6
selection, 3'->5' proofreading)
Accessory proteins (e.g. SSBP) ~10-7
Post-replication mismatch correction ~10-10
Mechanisms for maintaining genetic stability associated
with DNA replication in E. Coli

3. (a) Mismatches: Occurs
during DNA synthesis (i.e.
replication, repair, or
recombination)
Spontaneous alterations:

4. Nucleotides spontaneously under go a transient rearrangement of bonding, e.g. a
shift from NH2 (amino form) to NH (imino form) or C=O (keto) to C-OH
(enol). Therefore, if any base in a template strand exists in its rare tautomeric
form during DNA replication, misincorporation in the daughter strand can
result.

6. Three of the four bases normally present in DNA (cytosine, adenine, and
guanine) contain amino group (NH2). The loss of the amino group
(deamination) can occur spontaneously and result in the conversion of
the affected bases to uracil, hypoxanthine, and xanthine, respectively.

7. Depurination and depyrimidination:
The loss of purines or pyrimidines
from DNA usually occurs at
acidic pH; however, it can also
happen in physiological pH
(~10,000 purine per day in
mammalian cell; ~500
pyrimidine/day). This will results
in breaking the 3' phosphodiester
bond called b-elimination.

8. (a) Physical agents that damage DNA:
--- Ionizing radiation: OH, O2
-, H2O2, damage base and sugar
residues.
--- UV radiation: Cyclobutane pyrimidine dimers, Thymidine dimers
(T-T) dimer

9. (b) Chemical agents that damage DNA:
--- Alkylating agents: Alkylating agents are
electrophilic compounds with affinity for
nucleophilic centers in organic macromolecules.
These include a wide variety of chemicals, many of
which are proven or suspected carcinogens (such as
nitrous acid, hydroxylamine, and ethylmethane
sulfonate, EMS), Adding alkyl group to hydrogen-
bonding oxygen of G or T, resulting in G-T mispairing
G-C ---> G*T --->A-T
T-A --->T*-G ---> CG

10. A base analogue is a substance other
than a standard nucleic acid base
that can be incorporated into a
DNA molecule by the normal
process of polymerization. Such a
substance must be able to pair with
the base on the complementary
strand being copies, or the 3'->5'
editing function will remove it.
For example, 5-bromouracil is an
analogue of thymine and might
cause an A-T to G-C transition
mutation.

12. Intercalating agents: Substances whose dimensions are roughly the same as
those of a purine-pyrimidine pair. In aqueous solutions, these substances
form stacked arrays, and are also able to stack with a base-pair by insertion
between two base-pairs. This may result in frameshift mutation.

14. Chemicals that are metabolized to electrophilic reagents: Aflatoxins,
benzo[a]pyrene
A mutagen is a physical or chemical agent
that causes mutations to occurs.
Mutagenesis is the process of producing a
mutation.
Mutant refers to an organism or a gene that
is different from the normal or wild type.

15. Mutants may have second
mutation and become wild
type again.
Reversion was used as a
means of detecting
mutagens and carcinogens-
the Ames test

16. (1) Repair by direct reversal: The simplest mechanism. e.g. UV induced
T-T dimer is recognized by photolyase and is cleaved into intact
thymine (light dependent). This is called photoactivation

17. (2) Excision Repair: The most
ubiquitous repair mechanism,
which can deal with a large
variety of structural defects in
DNA.

18. (3) Recombinational repair (Postreplicational repair): Occurs before
excision repair has happened or when excision repair can not fix the
problem

19. (4) The SOS response: The SOS response system is only active in
response to some signal such as a blocked of replication fork. In E. Coli,
recA and lexA govern the expression of a number of other genes
involved in DNA repair. This is an error-prone DNA repair mechanism
and result in higher than normal mutagenesis.

20. 1. DNA damage
2. RecA converted to RecA*
3. RecA* facilitated LexA self-cleavage
4. Increased synthesis of SOS proteins
5. Error prone repair induced
6. DNA damage repaired
7. RecA* returned to RecA
8. LexA no longer self-cleaved
9. LexA repressed SOS genes
10. LexA repress lexA gene expression

21. Transition: One purine replaced by a different
purine;or one pyrimidine replaced by a
diferent pyrimidine
A G T C
Transversion: A purine replaced by a pyrimidine
or vice versa
I. Point mutation:
A. Base substitution
A T
Change in DNA
C G

22. Change in protein
1. Silent mutation: altered codon codes for the
same a.a.
2. Neutral mutation: altered codon codes for
functional similar a.a.
3. Missense mutation: altered codon codes for
different dissimilar a.a.
4. Nonsense mutation: altered codon becsomes
a stop codon
GAG (Glu) --->GAA (Glu)
GAG--->GAC (Asp)
GAG ---> AAG (Lys)
GAG ---> UAG (stop)

23. B. Frameshift mutation: addition or deletion of
one base-pair result in a shift of reading frame
and alter amino acid sequence
1. Wild type: ATG ACC AGG TC
2. Base addition: ATG ACA CAG GTC
3. Base deletion: ATG ACA GGT C
Arg
Met Thr
Met Thr Gln Val
Met Thr Gly

24. II. Insertion
III. Deletion
IV. Translocation
V. Inversion