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DNA Mutation and Diseases

Dr Anand Mohan

Define a Mutation
• A mutation is a stable change in DNA structure


Base sequence is changed

• Mutations are classified into several “types”

Molecular Nature of Mutation (what types of mutations arise)
• Point mutations (base substitutions): one base substituted for another • Transition mutations = A to G or C to T (purine to purine or pyrimidine to pyrimidine) • Transversion mutations = purine to pyrimidine or vice versa • Can arise from mispairing, insertion of base analogs, or chemical mutagens (nitrous acid, hydroxylamine and alkylating agents)

Consequences of Point mutations
• A missense mutation: This results in one wrong codon and one wrong amino acid.

Consequences of Point mutations
A nonsense mutation: If the change in the base sequence results in a stop codon, the protein would be terminated at that point in the message.

Consequences of Point mutations
• A sense mutation: This occurs when the change in the DNA sequence results in a new codon still coding for the same amino acid.

Other types of mutations
• Insertions and deletions: result in frameshift mutations (less common)
Insert a G here

DNA Mutation - Reasons
1) Through mistakes during DNA replication

2) Spontaneous mutations (deamination, depurination that occur naturally) 3) Induced mutations caused by environmental agents (chemical mutagens, UV radiation)

Spontaneous Mutagenesis
•Watson and Crick originally suggested that mutation could occur spontaneously during DNA replication if pairing errors occurred. •If a base of the DNA underwent a proton shift into one of its rare tautomeric forms (tautomeric shift) during the replication process, an inappropriate pairing of bases would occur. •Normally, adenine and cytosine are in the amino (NH2) form. Their tautomeric shifts are to the imino (NH) form. Similarly, guanine and thymine go from a keto (C=O) form to an enol (COH) form. •During DNA replication, a tautomeric shift in either the incoming base (substrate transition) or the base already in the strand (template transition) results in mispairing. •The mispairing will be permanent and result in a new base pair after an additional round of DNA replication. The original strand is unchanged

Adenine can pair with adenine if one of the bases undergoes a tautomeric shift while the other rotates about its base-sugar (glycosidic) bond. The normal configuration of the base is referred to as the anti configuration; the rotated form is the syn configuration. Since we now believe that as many as 10% of bases may be in the syn configuration at any moment, the transversion mutagenesis rate should be about 10% of the transition mutagenesis rate, a value not inconsistent with current information.

Some base-pair mutations can have serious results. If guanine undergoes an oxidation to 8-oxoguanine, it pairs with adenine. A GC base pair is converted to a TA base pair through an 8oxoguanineadenine intermediate. This transversion has been found to be common in cancers.

Base Analogs may get incorporated during DNA replication
Compounds that chemically resemble a nucleotide base closely enough that during DNA replication, they can be incorporated into the DNA in place of the natural base.

= thymine analog gets incorporated during DNA replication opposite an A

frequent tautomer change -now pairs with G

Another base analog
•The base analog, 2-AP pairs with T. •But 2-AP can also pair with C via a singe Hbond. •Therefore, if 2AP gets in DNA, then upon replication a C is sometimes inserted.

Spontaneous DNA Mutation
• Deamination: - C to U (now pairs with T) (100/cell/day) - A to Hypoxanthine (now pairs with C) - G to Xanthine (still pairs with C) • Depurination/Depyrimidination: - removes purine base from DNA(104 bases /cell/day) or pyrimidine bases (less frequent) • Oxygen radical damage -breaks sugar rings

Oxidative deamination
The primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to keto groups in reactions like the one pictured here:

In a mammalian cell, ~100 uracils are generated per cell per day in this fashion. Other reactions include conversion of adenine to hypoxanthine, and guanine to xanthine.

Oxidative deamination other examples

Spontaneous Base Loss
The glycosyl bond linking DNA bases with deoxyribose is labile under physiological conditions. Within a typical mammalian cell, several thousand purines and several hundred pyrimidines are spontaneously lost per cell per day.

Loss of a purine or pyrimidine base creates an apurinic/apyrimidinic (AP) site (also called an abasic site)

Oxygen radical damage

Mutation through Exposure to Environmental Agents

Chemical mutagens:

- Nitrous acid (HN02) formed from nitrites (in
preserved meats) reacting with stomach acid, causes oxidative deamination - Alkylating agents add methyl or ethyl groups to bases and change their pairing properties - Intercalating agents (ethidium bromide, acridine orange) distort the helix causing frameshift insertions or deletions.

Agents causing oxidative deamination

Nitrous acid


Agents causing alkylation

The first report of mutagenic action of a chemical was in 1942 by Charlotte Auerbach, who showed that nitrogen mustard (component of poisonous mustard gas used in World Wars I and II) could cause mutations in cells.

Intercalating Agents

Mutation through Exposure to Environmental Agents
• Ultraviolet Radiation: classified in terms of its wavelength
•UV-C (180-290 nm)--"germicidal"--most energetic and lethal, it is not found in sunlight because it is absorbed by the ozone layer •UV-B (290-320 nm)--major lethal/mutagenic fraction of sunlight •UV-A (320 nm--visible)--"near UV"--also has deleterious effects (primarily because it creates oxygen radicals) but it produces very few pyrimidine dimers.

Tanning beds have UV-A and UV-B.

Ultraviolet Radiation:

- causes dimers to form between adjacent T
bases on a strand of DNA (or between adjacent
T-C bases, hence the term pyrimidine dimer)

- a cyclobutyl ring links the adjacent T’s
- this interferes with the ability of the T’s to base pair to the opposite strand, and blocks DNA replication

The most frequent photoproducts are bonds formed between adjacent pyrimidines within one strand The most frequent are CPDs, cyclobutane pyrimidine dimers T-T CPDs are formed most readily, followed by T-C or C-T; C-C dimers are least abundant. CPDs cause a distortion in the DNA chain structure. The two adjacent pyrimidines are pulled closer to each other than in normal DNA.

T-T CPD with cyclobutyl ring in blue

Nucleotide Excision Repair defects in Humans Xeroderma pigmentosum (XP)
Genetic disorder with symptoms: -extreme sensitivity to sunlight (by ~age 2), and >1000X higher risk of skin cancer (by ~age 8) Defect is in repair of UV damage
Gene mapping identified several repair proteins (called XP proteins) XP-C and XP-A recognize pyrimidine dimers XP-B and XP-D have helicase activity XP-G and XP-F have nuclease activity

Real-world biochemistry
In 1997, NASA developed a Prototype UV garment for children with XP, Porphyria and other sun Related Disorders to have a Quality of life and Freedom. This NASA UV Protective Project designed for XP was completed in 1998 and the UV garments are being supplied to children of parents that have requested them.

Sickle cell disease
•Haemoglobin is made up of 4 polypeptide chains (2 alpha & 2 beta chains). •Haemoglobin is made up of 4 polypeptide chains (2 alpha & 2 beta chains). •Gene for beta chain is found on chromosome 11 and consists of 438 bases. •Gene for beta chain is found on chromosome 11 and consists of 438 bases. •A mutation occurs in the gene coding for the beta chain. •A mutation occurs in the gene coding for the beta chain. •The mutation is a substitution where adenine replace thymine on the DNA •The mutation is a substitution where adenine replace thymine on the DNA template strand. template strand. •As a result the amino acid valine replaces glutamic acid. •As a result the amino acid valine replaces glutamic acid. •This change the properties of the haemoglobin and results in distorted red •This change the properties of the haemoglobin and results in distorted blood cells. blood cells. haemoglobin normal red blood cell sickle cell HBB gene on chromosome 11

This single point mutation has a dramatic effect. Individuals have many health problems, eg weakness, jaundice, anaemia, heart & kidney defects, brain damage, skin lesions and inflamed spleen.