PHAR2811 Dale’s lecture 6
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Phar lecture 6: DNA repair and Mutation
Repair :
Repair of DNA is vital to the survival of the species. DNA has the capability to repair itself, unlike
RNA. The extra copy provides the template and elaborate repair mechanisms have evolved to correct
corruptions. Many errors at the time of replication are corrected by the 3’  5’ exonuclease activity of
DNA pols I & III.
Apart from these there are corruptions to the sequence which occur after replication. These are dealt
with by the various repair mechanisms. An example from Voet and Voet. There are 3.2 X 109 purine
nucleotides in the human genome. Each day ~10 000 glycosidic bonds are cleaved from these purines
in a given cell under physiological conditions. The conclusion: your cells contain some nasty little
compounds. There are 130 genes which encode proteins responsible for repair in the human genome.
Even lower life forms (E. coli, younger siblings etc) have a large range of repair mechanisms. Let’s
consider a few examples:
UV damage (or the battle of the bulge)
DNA, when exposed to UV irradiation, forms pyrimidine dimers. Carbons 5 and 6 of two consecutive
pyrimidines form covalent bonds with each other (cyclobutyl ring). This happens most often with
ajoining thymine nucleotides. These cyclobutyl bonds are ~1.6 Å which is much shorter than the
distance between the bases (~3.4 Å. This causes a bulge in the double helix which prevents
transcription or replication. This dimer has to go!!
There are a couple of ways of repairing this dimer. One is to cleave the bonds directly, with an
enzyme known as photolyase. This enzyme contains a chromophore which absorbs light in the 300 –
500 nm region. Once excited it transfers this trapped energy to a second molecule (FADH-) which
donates an electron to the dimer bond. This splits the dimer. The nucleotide with the extra electron
then reduces the FADH back to FADH-. Photolyase is present in many prokaryotes and eukaryotes
(not humans however).
Excision repair.
Another more general type of repair is excision repair. The pyrimidine dimer, and a number of
ajoining nucleotides are cut out by a set of enzymes responsible for nucleotide excision repair (NER).
NER is carried out in E. coli by UvrA, UvrB and UvrC. These enzymes seek out the bulge and cut out
the offending nucleotides. The 11 to 12 oligomer is then removed by UvrD. DNA pol I then comes in
fills in the missing section, using the other strand as a template. The gap is sealed by DNA ligase.
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Uracils in DNA
Uracil, which comes about from the spontaneous deamination of cytosine or for that matter
hypoxanthine (another base which comes about from the deamination of adenine) and xanthine
(derived from the deamination of guanine), does not belong in DNA. A set of enzymes (base excision
repair, BER) cleaves out the base at the glycosidic bond leaving an apurinic or apyrimidinic site (AP).
The deoxyribose is then excised by an AP endonuclease along with several ajoining nucleotides. The
whole section is then filled in by DNA pol I and DNA ligase. When purine bases are cleaved at the
glycosidic bond (as in the example in the introduction) the AP endonuclease again comes into action
to remove the deoxyribose. The DNA polI and ligase then mop up.
Mutations
A mutant gene is one that has a different sequence to the normal or wild type gene. This change is
inheritable. There are a variety of mutations which may or may not cause a change in the phenotype.
The vast majority of mutations are neutral, having no effect (positive or negative) on the organism.
Types of mutations
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Transversion: purine replaced by a pyrimidine or vice versa. This has implications for the 3-D
shape of the helix.
Transition: pyrimidine for pyrimidine or purine for purine e.g. AG,CT
Silent mutations: altered codon still codes for the same amino acid (code redundancy)
Frameshift: shifts the reading frame by adding or deleting base(s). Leads to a non-functional
protein.
Neutral mutation: altered codon codes for a functional similar amino acid  no effect on the
functionality of protein.
Point mutation: single base pair change, can be a substitution, deletion, or addition.
Missense: altered codon for functionally different amino acid  these can be lethal
Nonsense: mutation produces a stop codon  truncated protein  also dangerous.
Splice mutation: produces a splice site or removes one (only in eukaryotes)
Temperature sensitive: mutation causes a change the protein function which is temperature
sensitive. Usually the protein functions normally at lower permissible temperatures (<30oC) but is
inactive at higher temperatures (>40oC).
Reversion: a mutation which reverts to wild type, referred to as revertants.
Leaky mutation: A mutation which doesn’t affect the organism under normal conditions. It will
show up in “stressed” conditions.
Mutagenesis.
Definition of mutagen: a physical or chemical agent which causes mutation to occur at a higher
frequency.
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Natural or spontaneous mutations: These are the mutations which occur at a normal background
rate all the time. These mutations in the genome can arise naturally in the course of a cell’s life by the
following mechanisms:
 The alternative tautomer of the base. If the base flips to the alternative tautomer at the time of
replication the wrong nucleotide will pair up and by two rounds of replication we have a base pair
switch.
 An error in replication which is not picked up by the proof reading activity of DNA pol III will
result in a mutation.
 Deamination, of cytosines (discussed earlier) or adenine to hypoxanthine. These can be corrected
by specific repair mechanisms.
 Depurination (see above) can also be corrected by specific repair mechanisms.
Induced mutagenesis: Chemicals (synthetic), which increase the frequency of mutagenesis.
 Intercalators, planar ring structures which slide in between the base pairs causing a disruption to
the normal base stacking eg ethidium Bromide, acridine orange, actinomycin D.
 Alkylating agents, which methylate or ethylate bases and result in altered base pairing during
replication e.g methylmethane sulfonate (MMS), nitrosamine.
 Base altering agents eg nitrous acid converts amino groups to keto groups.
 Base analogues e.g. 5-Bromouracil which replace thymine but base pair to G (tautomeric shift
due to Br).
Ames test for mutagens.
There are an increasing number of possible carcinogenic (cancer causing) chemicals in our
environment. Carcinogenic compounds are often mutagenic; the mutation caused often leads to
uncontrolled cell proliferation. The Ames test is a simple test for potential mutagens, which relies on a
strain of Salmonella which is His- . This strain is grown on a plate containing minimal histidine (just
enough for maintenance not growth). A disc with the test mutagen is placed on a disc in the center of
the plate. If the compound does cause mutations it will increase the rate of reversion i.e. His- mutants
reverting to His+. These revertants will be able to grow and divide on the minimal His medium and
show up as a colony. You simply count the colonies near the disc. The number of colonies is usually
proportional to the dose of the potential carcinogen.
Sometimes synthetic chemicals are not mutagenic until metabolized by our body. The P450 system
operating in our liver to detoxify natural foreign compounds can actually convert some of these
synthetic chemicals to mutagens by enzymatic modification. A liver microsomal fraction is
incorporated into the medium to check for in vivo conversion.