Classes of Mutations mutations 2 Frameshift 3 Gross

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

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

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

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.

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).

The strains… Wild-type strain -- provides reference for frequency of mutation when repair and coping systems are intact. Excision repair mutant (uvr. A) -- unable to remove DNA lesions. Mutant in damage-inducible DNA polymerase (umu. C) -umu. C allows mutagenic bypass of UV lesions. Mismatch repair mutant (mut. S) -- impairs removal of mismatched base pairs that have passed through proofreading step of DNA replication

UV irradiation and plating protocol

Pull-down Assay