1. Classes of Mutations 1. Base-pair changes (transitions & transversions) 3. Gross rearrangements 2. Frameshift mutations ^

2. Consequences of mutations Base substitutions lead to nonsense, missense, neutral or silent mutations Frameshift mutations alter the translational reading frame Rearrangements reduce or eliminate protein function

3. Methods for Inducing Mutagenesis Protonated 2AP cytosine 2APthymine Base analogs Alkylating agents SOS-dependent Mutagens (e.g. UV light, Aflatoxin B 1 )

4. Methods for Detecting Mutagenesis Variety of test systems available that are based on acquisition or loss of a specific genetic marker, e.g. antibiotic resistance.

5. UV-induced mutagenesis (using rif-resistance) Score for rif S to rif R. Rifampin (rif) targets the bacterial RNA Pol, blocking transcription. 69 possible single base substitutions confer rif resistance. These are concentrated in the first half of the protein, and are distributed among 24 coding positions (Garibyan et al. DNA Repair 2 (2003) 593-608).

6. The strains… Wild-type strain -- provides reference for frequency of mutation when repair and coping systems are intact. Excision repair mutant (uvrA) -- unable to remove DNA lesions. Mutant in damage-inducible DNA polymerase (umuC) -- umuC allows mutagenic bypass of UV lesions. Mismatch repair mutant (mutS) -- impairs removal of mismatched base pairs that have passed through proof- reading step of DNA replication

7. UV irradiation and plating protocol

8. Pull-down Assay