1. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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 O pen R eading F rame (ORF) sequence. AAT DNA UUA mRNA Leu amino acid CUA GUA AUA UCA UUC UUG UUU UCA Leu Val Ile Ser Phe Leu Phe Ser UGA UAA Stop Can mutate to…..

2. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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. 3’ 5’ 3’ Gap maybe up to a few 1000 bps Replication slippage Recombination and deletion

4. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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. CAG Gln UAG STOP Amber GUC Gln tRNA AUC Mutation in another tRNA leads to incorporation of a different amino acid Protein may or may or be functional

5. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. F. Mechanisms of mutation. Cont…. N N O CH 3 H H H O 2 x H Bonds Thymine in its KETO form Will bind to Adenine TA pairing as expected during replication N N O CH 3 H H O 3 x H Bonds Thymine in its ENOL form. Binds Guianine. TG pairing during replication H

7. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. F. Mechanisms of mutation. Cont…. Adenine in its AMINO form Will bind to Thymine. TA pairing as expected. Adenine in its IMINO form. Binds Cytosine. AC pairing. Still 2 x H Bonds N NH N H 2 x H Bonds N NH 2 N SIMILARLY Forms in equilibrium with about 1 in 10 4 or 10 5 molecules in the ENOL or IMINO forms. Therefore natural transition per base per generation is also about 1 in 10 4 to 10 5.

8. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. F. Mechanisms of mutation. Cont…. Must also have segregation after replication ATAT GTGT GCGC ATAT 1st replication 2nd replication This would happen about 1 in 10 4 to 10 5 ! The potential for many deleterious mutations is high i.e. GENETIC LOAD is too high  Dale Ch 3

9. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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 UV 254nm ) Repair of this damage rapidly can lead to mutagenic effects. F. Mechanisms of mutation. Cont….  Dale Ch 3

10. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. 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 F. Mechanisms of mutation. Cont….

11. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. AG or CT Mismatch Polymerase Removal of mismatch Polymerisation in 5’ - 3’ direction 3’ - 5’ digestion by polymerase F. Mechanisms of mutation. Cont…. Proofreading carried out by the 3’ to 5’ exonuclease activity of the DNA Polymerases III and I. THIS WORKS AT ONE LEVEL