Isolation of Mutants; Selections, Screens and Enrichments Carolyn Keeton Turn In HW 1 in Front

Outline What causes mutations? Spontaneous Mutator Strains Mutagens Considerations Isolation of Mutants Selections Screens Enrichments

Transitions vs. Transversions Pu -> Pu Py -> Py G -> A C -> T Transversions Pu -> Py Py -> Pu G -> T C -> A G -> C C -> G A, G Pu T, C Py

What causes mutations? Spontaneous- wide variety of mutations types substitutions, deletions, frameshifts, insertions DNA replications errors- not repaired Recombination –> rearrangements-> deletions and insertions (duplications) DNA damage – radiation, metabolisms, free radicals Transposable elements – insertions, usually rare, <106/gene/ generation

Types of Mutations Missense Insertion Deletion Frameshift

Mutator strains Cells have mutation that affects DNA repair and metabolism (not WT) Examples: mutD = dnaQ= proofreading subunit of DNA Pol III -Mutation rate increases 1000x Substitutions, transitions, transversions, and frameshifts mut L S H= mismatch repair Mutation rate increases 100x 3. Several others – metabolism and repair Repair Mutation

Mutagens Chemical or physical agents Increase mutation frequency How do they work? 1. Mispairing 2. Modify bases in DNA

Types of mutations produced Types of Mutagens Mutagen Mechanism Types of mutations produced Spontaneous DNA replication and repair errors, spontaneous modification of nucleotides All types of mutations produced UV irradiation Pyrimidine dimers induce error prone repair (SOS) Mainly G-C to A-T transitions, but all other types of mutations including deletions, frameshifts, and rearrangements at somewhat lower frequency 2-aminopurine (2AP) Base analog A-T to G-C and G-C to A-T transitions Bromouracil G-C to A-T and A-T to G-C transitions Hydroxylamine (NH2OH) Alkylating agent, generates N4-hudroxycytosine G-C to A-T transitions when used in vitro N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) Alkylating agent, generates O6-methylguanine G-C to A-T transitions, multiple, closely spaced mutations common Ethylmethane sulfonate (EMS) (EMS) G-C to A-T transitions Ethylethane sulfonate (DES) Alkylating agent, induces SOS response G-C to T-A transversions, other base substitution mutations Nitrous acid Oxidative deamination G-C to A-T and A-T to G-C transitions, deletions produced at a lower frequency ICR-191 Intercalating agent, alkylacridine derivative that stabilizes looped out bases by stacking between them Frameshifts, mainly additions or deletions in runs of G or C

Mispairing Occurs during replication by incorporating the wrong base Cells must be actively growing for these to work Example 5BU (Thymine Analog), or 2AP (Adenine analog)

5BU

Hydroxylamine

Modify bases in DNA By mispairing Can use alkylating agents Ex. Diethylsulphate, methylates guanine to pair with thymine during replication= results in GC to an AT bp Ex. Nitrosogunidine is very potent (can’t buy anymore) Thought to act at the replication fork Ex. Depurination induces SOS repair

HA Interacts directly with the DNA by modifying the base Modifies C to bp with an A instead of a G Specific for GC to AT Only works in vitro, no true revertants as unidirectional We have sequenced hundreds of mutants, only 1 was not GC to AT

Altered nucleotide pools Alters concentration of nucleotides, increases rate of misincorporation Exc BrUTP inhibits dCTP synthesis Increases T to G misincorporations Modifying the nucleotide pools = increases rate of misincorporations Ex. PCR dNTP concentrations

Intercalating Agents Insert between bases in DNA Causes frameshifts Ex EtBR- carcinogen

Indirect Mutagenesis Occurs during repair of DNA damage Induce SOS pathway Ex. Expose to UV light or MMS (alkylation) Makes T-T dimers which block replication Induces repair High probability of a mutation during DNA synthesis past lesion Results in substitutions, rearrangements. and frameshifts

Types of mutations produced Types of Mutagens Mutagen Mechanism Types of mutations produced Spontaneous DNA replication and repair errors, spontaneous modification of nucleotides All types of mutations produced UV irradiation Pyrimidine dimers induce error prone repair (SOS) Mainly G-C to A-T transitions, but all other types of mutations including deletions, frameshifts, and rearrangements at somewhat lower frequency 2-aminopurine (2AP) Base analog A-T to G-C and G-C to A-T transitions Bromouracil G-C to A-T and A-T to G-C transitions Hydroxylamine (NH2OH) Alkylating agent, generates N4-hudroxycytosine G-C to A-T transitions when used in vitro N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) Alkylating agent, generates O6-methylguanine G-C to A-T transitions, multiple, closely spaced mutations common Ethylmethane sulfonate (EMS) (EMS) G-C to A-T transitions Ethylethane sulfonate (DES) Alkylating agent, induces SOS response G-C to T-A transversions, other base substitution mutations Nitrous acid Oxidative deamination G-C to A-T and A-T to G-C transitions, deletions produced at a lower frequency ICR-191 Intercalating agent, alkylacridine derivative that stabilizes looped out bases by stacking between them Frameshifts, mainly additions or deletions in runs of G or C

How specific are mutagens? J. Miller had a collection of lacI amber mutants- examine reversion to lacI+ EMS, NG, 2AP, UV = several sites in common UV mostly changes at other sites mutT different pattern: transversion 2AP had hot spots (regions prone to mutagenesis) Handout

Some Genes do not mutagenize well Ex rII gene of phage T4 Benzer isolated 3000 spontanous mutants ½ were at 2 sites Need to do lots of work to isolate new mutants

Choice of Mutagens NG: very powerful: initial test to see if you can isolate mutants ICR: Only makes frameshifts HA: Only specific in vitro No true revertants (for HA) (Explain later)

Insertional Mutants Use a transposon with an antibiotic cassette Inserts into chromosomes Isolate mutant Can sequence the adjacent DNA to determine what was disrupted Good for finding undiscovered targets

Zen of Mutagenesis Should I isolate a spontaneous mutant or use a mutagen? Assumptions (generous) 1 gene = 103 basepairs= 330 amino acids Chromosome = 5 x 106 bp for E. coli Assume 1. Equal random change to mutate any bp 2. Assume 1 hit/cell 103 bp = 1/5000 probability of hit in a gene 5x 106 bp Assume 10% = phenotype (90% silent) 1 1 1 5000 10 5x104 If screened 250 colonies a plate= 200 plates for 1 mutant (Lots of work) If use a mutagen that increases mutation rate 100x= 2 plates (but can get second site mutants) x =

*Backcross* Very important when doing mutagenesis After isolating a mutant, a good geneticist would move it to a clean background Verifies the mutation gives the correct phenotype Will explain the mechanism later x x x x x x x x