D. Types of mutation

1. D. Types of mutation • 1. Point mutation. • Affecting non coding regions: e.g. Promoter/operator • e.g. -10 TATAAT sequence in promoter • Affecting CODING SEQUENCE or Open Reading Frame (ORF) sequence. AAT DNA UUA mRNA Leu amino acid Can mutate to….. CUA GUA AUA UCA UUC UUG UUU UCA Leu Val Ile Ser Phe Leu Phe Ser UGA UAA Stop Stop

2. D. Types of mutation. Cont……. • STOP leads to truncated protein and termination of translation • (Note: Transcription termination involves inverted repeats and role of rho protein factor) • Some codons are NONSENSE (i.e. normally no tRNA) • UAG Amber (after discoverer Bernstein :German for Amber) • UAA Ochre • UGA Opal • 2. Frameshifts. Addition or deletion of bases leading to altered sequence beyond the sequence change.

3. 3’ 5’ 3’ 5’ Gap maybe up to a few 1000 bps D. Types of mutation. Cont……. • 3. Deletions • Arise spontaneously where there are direct repeats of over 4-5 base pairs. More frequent when there is more sequence repeated (see later excision of Transposons and IS elements) i.e. Replication slippage Recombination and deletion

4. GUC Gln tRNA E. Reversion and Suppression • Back mutation to original sequence is rare • Suppressor mutations relieving mutations within coding regions more common • Leads to insertion of amino acid at stop codon e.g. UAG STOP Amber CAG Gln AUC Mutation in another tRNA leads to incorporation of a different amino acid Protein may or may or be functional

5. F. Mechanisms of mutation • Spontaneous mutation. ERRORS IN REPLICATION • Usually only AT GC pairing allowed • Repair systems are present • How do mis-matches or mis-pairings happen ? • Alternative Tautomeric forms of A and T occur • (“In evolution we are all prisoners of simple chemistry”)  Dale Ch 3

6. O H CH3 N N H O H F. Mechanisms of mutation. Cont…. 2 x H Bonds H 3 x H Bonds O CH3 N N H O H Thymine in its ENOL form. Binds Guianine. TG pairing during replication Thymine in its KETO form Will bind to Adenine TA pairing as expected during replication

7. NH NH2 H N N N N F. Mechanisms of mutation. Cont…. SIMILARLY Still 2 x H Bonds 2 x H Bonds Adenine in its IMINO form. Binds Cytosine. AC pairing. Adenine in its AMINO form Will bind to Thymine. TA pairing as expected. Forms in equilibrium with about 1 in 104 or 105 molecules in the ENOL or IMINO forms. Therefore natural transition per base per generation is also about 1 in 104 to 105.

8. Must also have segregation after replication 2nd replication 1st replication G C G T A T A T F. Mechanisms of mutation. Cont…. This would happen about 1 in 104 to 105 ! The potential for many deleterious mutations is high i.e. GENETIC LOAD is too high  Dale Ch 3

9. F. Mechanisms of mutation. Cont…. • WORKING AT ANOTHER LEVEL AFTER REPLICATION IS Mismatch repair. • A form of excision repair (see later). DNA adenine methylation is involved in recognising sections of DNA to be repaired after replication. • DNA can also become damaged due to a variety of influences. • Chemical mutagens • Ionising radiation (such as X rays,  rays and UV254nm) • Repair of this damage rapidly can lead to mutagenic effects.  Dale Ch 3

10. F. Mechanisms of mutation. Cont…. • Chemical mutagens • Base analogues: substitute for normal base but are not proofread - mispairing • 2-aminopurine: Adenine analogue which pairs with cytosine • 5-bromouracil: thymine analogue pairs with guanine • Modification of bases: Do not require replication and induce DNA repair. • Nitrous acid (deaminates); hydroxylamine (reacts with cytosine) • Alkalyating agents transfer alkyl groups to bases. Very powerful; ethyl methane sulphonate (EMS); N-methyl, N-nitrosoguanidine (NTG) • Intercalating agents: additions or deletions caused • acridine orange; ethidium bromide

11. 3’ - 5’ digestion by polymerase AG or CT Mismatch Polymerase F. Mechanisms of mutation. Cont…. Proofreading carried out by the 3’ to 5’ exonuclease activity of the DNA Polymerases III and I. Removal of mismatch Polymerisation in 5’ - 3’ direction THIS WORKS AT ONE LEVEL