Lecture #22

Thursday November 29, 2001

Mutations and DNA Repair Mechanisms (Ch 15 pp 398-410; Ch 14)

1. PROTEINS AS END PRODUCTS OF GENES

· Original concept of one gene : one enzyme

- based on studying inborn errors of metabolism

- Beadle and Tatum proved theory correct studying Neurospora nutritional mutants caused by the loss of activity of one enzyme

- But many proteins made up of >1 polypeptide!

- Now modified to be one gene : one polypeptide chain

§ Proteins have complex structure subject to mutation in different ways

§ Different types of proteins encoded by genes and mutations cause different phenotypic effects

- enzymes

- transport proteins

- regulatory proteins

- structural proteins

2. MUTATIONS

§ Defined as heritable changes in genetic material

- chromosomal mutations vs ntd sequence mutations

· Gametic vs somatic cell mutations

- Mutations in germ-line cells are heritable.

- Mutations in somatic cells not heritable, except in plants.

· Varying phenotypic effects of mutations

- Morphological –

- Biochemical –

- Behavioral –

- Regulatory –

- Conditional –

· Spontaneous mutations

- Random changes in ntd sequence

- Usually due to defects during replication

- Measurable rate

- Forward mutations happen more frequently than reverse mutations

- Neutral vs deleterious

· Induced mutations

- Caused by exposure to agent

- “Natural” outside agent vs experimentally applied vs environmentally induced

- Measurable rate may be increased due to induced mutations

3. MOLECULAR BASIS OF MUTATIONS

· Single base substitutions = point mutations

- Transitions – purines switch or pyrmidines switch

- Transversions – purines and prymidines switch with each other

· Consequences of point mutations

- Silent – amino acid does not change

- Missense - amino acid does change

q May be deleterious if biological properties of peptide are changed.

q May have no effect if new amino acid has same functional characteristics

- Nonsense – a stop codon is prematurely encoded instead of an amino acid

· Insertions and deletions of nucleotide sequences

- Adds a single or group of nucleotides

- Cause a frameshift mutation

4. SPECIFIC EXAMPLES of mutations and their effects on phenotype (in humans)

5. MECHANISM OF MUTATION

· Tautomeric shifts

- transient shifts in chemical form may trigger mispairing during replication

· Chemical mutagens

- Base analogues

q Chemical structure almost identical to nucleotide

q e.g. 5-BU very similar to T but can pair with A or G will result in a transition after replication

- Hydroxylating agents

q Add OH group to base

q e.g. Hydroxylated C now pairs with A

- Alkylating agent

q Add an akyl group (ethyl or methyl)

q e.g. Mustard gas, EMS

- Deaminating agent

q Removes amino group from the nucleotide

q e.g. nitrous acid modifies C to U (now pairs with A) and modifies A to hypozyanthine (now pairs with C)

- Intercalating agents

q Inserts in between base pairs causing conformational change in double helix

q Causes frameshift mutations due to insertions or deletions of a few nucleotides

q e.g. Acridine dyes, DAPI

· Radiation

- Ionizing radiation

q x-rays, gamma rays, cosmic rays

q produce highly reactive ions called free radicals which can break chromosomes, alter bases, cause strand breakage

- Nonionizing radiation – UV light

q Changes bases by forming pyrmidine dimers

6. DNA REPAIR

· DNA polymerase proofreading function

- Nucleotide mismatch creates bulge in DNA

- Exonuclease activity cuts out mismatch

· Photoreactive repair in bacteria using photolyase – cleaves double TT bonds caused by UV light dimerization

§ Excision repair

- Nuclease cuts out mismatched base

- DNA polymerase adds nucleotide

- DNA ligase seals gap

· Methyl-directed mismatch repair

- If proofreading function fails……

- Parental strands are marked with methyl groups

- Enzyme recognizes mismatch in replicated DNA (unmethylated strand)

- Wrong DNA is excised.

- Newly synthesized (and corrected strand) is methylated

· Double strand break repair

- If both strands are nicked, protein complex will repair nicks in correct place

· SOS repair system in bacteria

7. EXAMPLES of mutations in DNA repair system

· Xeroderma pigmentosum

· Bloom’s syndrome

· Fanconi’s syndrome