Mutation The process that produces a gene or a chromosome set that is differing than that of the wild type. The gene or a chromosome set that results from such a process.

Pairing between the normal (keto) forms of the bases Mismatched bases. Rare tautomeric forms of bases result in mismatches

Transition

MUTAGENS Radiation Chemical Agents Mobile Genetic Elements

Mutagens Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

2-AP: 2- aminopurine Analog of adenine that can pair with cytosine in its protonated state Normal pairing 14

2-AP: 2- aminopurine Analog of adenine that can pair with cytosine in its protonated state Normal pairing

5-BU :5 bromouracil An analogue of thymine 5-BU can be mistakenly incorporated into DNA as a base. The ionized form base pairs with guanine. Normal pairing

Transition

Rare form of BrU in Template

Mutagens מוטגנים Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

Alkylation-induced specific mispairing. Treatment with EMS alters the structure of guanine and thymine and leads to mispairings Transition

Alkelating agents

A powerful carcinogen originally isolated from peanuts infected with fungus. Alfatoxin attaches to guanine at the N-7 position. This leads to the breakage of the bond between the base and the sugar, thereby liberating the base and resulting in an apurinic site. Agents that cause depurination at guanine residues should tend to induce GC to TA transversions

Usually an A is inserted instead of the depurinated site

2-AP EMS NG

Mutation rate, a question of balance

MutationsDNA repair

Mutagens Chemical Agents Base analogs Base modifying agents Intercalators Other classes Millions of natural and synthetic compounds

Flat planar molecules that mimic base pairs and are able to slip themselves in (intercalate) between the stacked nitrogen bases at the core of the DNA double helix. In this intercalated position, an agent can cause single-nucleotide-pair insertions or deletions Intercalators

DAPIhCia2-GFPα-Tub1Merge Metaphase Telophase Cytokinesis TelophaseAnaphaseMetaphase CIA knock-down Control TelophaseAnaphaseMetaphase DAPI α-Tub1 Merge Anaphase Telophase Metaphase

Mutagens Radiation Chemical Agents Mobile Genetic Elements Ultraviolet (UV) Ionizing

UV light generates photoproducts Occurs between two adjacent pyrimidines on the same DNA strand The UV photoproducts significantly perturb the local structure of the double helix. These lesions interfere with normal base pairing. The C to T transition is the most frequent mutation, but UV light also induces other base substitutions (transversions) and frameshifts, as well as larger duplications and deletions. Pyrimidine: T, C

Transition Transversion

Mutagenes induce mutations by a variety of mechanisms. Some mutagenes mimic normal bases and are incorporated into DNA, where they can mispair. Others damage bases, which then are not correctly recognized by DNA polymerase during replication, resulting in mispairing MESSAGE

The Ames test A method that uses bacteria to test whether a given chemical can cause cancer. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound Bruce Ames 1928-today University of California, Berkely

TA100- sensitive for reversion by base pair substitution TA 1535/8 frameshift Ames test Strains inactive for BER and prone for Entry of molecules

Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the mutagenic potential of metabolites formed by a parent molecule in the hepatic system can be assessed

Genetic screens based on random mutagenesis have been seminal in identifying genes involved in biological processes such as genome stability

Yeast: A model eukaryote Yeasts – the ultimate model eukaryote for unicellular issues and some basic cell-cell interactions Yeast studies have broken new ground in: Cytoskeleton functionstranscription mechanisms** cell cycle**transcriptional regulation organelle biogenesischromatin modification secretion*signal transduction protein targeting mechanismsprotein degradation* chromosome replicationDNA repair genome dynamicsretroviral packaging prionsrecombination mechanisms ageingfunction of new genes metabolismprotein modification *Lasker Award **Nobel Prize "for their discoveries of key regulators of the cell cycle" Lee HartwaellPaul NurseTim Hunt The Nobel Prize in Physiology or Medicine 2001 for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells James E. RothmanThomas C. SüdhofRandy W. Schekman The Nobel Prize in Physiology or Medicine

One of the major approaches that led to the understanding of cell cycle regulation was the genetic analysis of yeasts, was pioneered by Lee Hartwell and his colleagues in the early 1970s. These investigators identified temperature-sensitive mutants that were defective in cell cycle progression. The key characteristic of these mutants (called cdc for cell division cycle mutants) was that they underwent growth arrest at specific points in the cell cycle. cdc28 caused the cell cycle to arrest at START, indicating that the Cdc28 protein is required for passage through this critical regulatory point in G 1

Yeast secretory pathway Thin-section electron micrographs of SEC1 mutant cells grown at the permissive temperature (left) and restrictive temperature (right).

The random mutagenesis approach rarely achieves saturation, because mutability, resulting in haploid viable mutants, varies widely among genes The best way to determine the whole spectrum of genes involved in a certain phenotype is to systematically analyze each of them The best way to determine the whole spectrum of genes involved in a certain phenotype is to systematically analyze each of them

DELETION LIBRARY Availability: available commercially Availability: available commercially Description: collection of strains deleted in all the non-essential genes. Description: collection of strains deleted in all the non-essential genes. Creation: replacing the gene in question by barcod containing selectable marker based on homologus recombination Creation: replacing the gene in question by barcod containing selectable marker based on homologus recombination gene1::KmX KanMX4 GENE1 gene1::KmXgene2::KmXgene3::KmXgene4::KmXgene4700::KmX KmX is a gene that provides resistance to the drug G418

Uptag U1 U2 D2 D1 Downtag xxx  ::kanMX4 yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX

2 main ways of studying the knockout collection: 1.Plate-based 2.Chip-based

Serial analysis of deletion strains (plate based) Apply Selection Identify deletion strains with growth defects ,000

each deletion strain in quadruplicate No Selection + Selection

One experiment results in the whole spectrum of genes that play a role in the repair of DNA damage One experiment results in the whole spectrum of genes that play a role in the repair of DNA damage

Large-scale Mapping of Genetic and Interactions in Yeast Synthetic Lethal Genetic Interaction in Yeast

Synthetic Lethality yfg1 yfg2 Dead Viable yfg1 yfg2

Synthetic Lethality A B aa B X A bb ViableLethal aa bb Wild-type Viable X X X Functional Relationships Essential biological function SL interaction Pathway B A2 A3 B1 B2 B3 Pathway A A1 A2 A3 B1B2B3 Complex AComplex B

How can we detect synthetic lethal interactions between a mutant of Interest (query gene) and other genes How can we detect synthetic lethal interactions between a mutant of Interest (query gene) and other genes

B Mating Heterozygous diploid Tetrad BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Dissection Meiosis a b A B a b A B a b A B a b A

Yeast tetrad analysis (classic method) tetrad Step1: separate spores by micromanipulation with a glass needle Step2: place the four spores from each tetrad in a row on an agar plate Step3: let the spores grow into colonies

Classical approach (tetrad dissection) BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Tetrad Dissection bni1∆ bnr1∆

We ask which mutation is synthetically lethal with our query mutant yfg1::ClonNAT Mating yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX Query gene Meiosis 4700 heterozygous diploids Tetrads The problem is that it is impossible to perform 4700 tetrad dissections wt

NPD PDT TT a b A B a b A B MATa MATα MATa a b A B MATα MATa a B b A a B MATα MATa b A A B a b

yfg1::ClonNAT yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX Query gene Mating 4700 heterozygous diploids Meiosis Tetrads We ask which mutations are synthetically lethal with our query mutant The SGA approach allows to do it in a systematic way We ask which mutations are synthetically lethal with our query mutant The SGA approach allows to do it in a systematic way Among the many tetrads only the MATa haploid double mutants spores can be selected yfg1::KmXyfg2::KmXyfg3::KmXyfg4::KmXyfg4700::KmX yfg1::ClonNAT Use the haploid selection marker for selection

Multiplicative Model Expected Double Mutant Fitness wt Fitness “Neutral” Expected Result, Multiplicative Model (additive) 0.25 aa bb ab  R. Mani F. Roth et. al., PNAS 2008 Mar 4;105(9): Epub 2008 Feb 27 D. Segre, R. Kishony et al., Nature Genetics 37, (2004) S. R. Collins, N. Krogan, J. Weissman, Genome Biol 2006;7:R63. “S-Score””

Two Basic Types of Genetic Interactions wt “Negative” Synthetic Lethal “Neutral” Expected Result aa bb ab  Fitness (colony size) 1

Two Basic Types of Genetic Interactions wt “Neutral” Expected Result 0.5 “Positive” e.g. two genes whose products are in the same nonessential complex/pathway Epistasis aa bb ab  FitnessFitness (colony size) 0.5 Protein complex A B C Non-functional B C A C C

Synthetic lethal interactions Conditional alleles of sec13 and sec23 show synthetic lethality at low temperatures

Genes that act in the same pathway (or complex) will share the same synthetic lethal interactions A complete map of all possible genetic interactions has the potential to reveal all pathways and complexes!

SGA Screening Lab (University of Toronto)

Pairwise genetic interactions can be represented by a graph 8 SGA Screens: 291 Interactions 204 Genes 8 SGA Screens: 291 Interactions 204 Genes

Genetic Interaction Network 132 Screens 4000 Interactions 1000 Genes ~200,000 Interactions/genome Amy Tong, Fritz Roth et al., Science 303: (2004)

Tong et al., Science 303: Global Mapping of the Yeast Genetic Interaction Network

All dynein subunits are SL with the same subset of mutants YMR299c is probably a new component of the dynein complex dynein SGA analysis clusters related genes Array gene clusters Query gene clusters

~2000 Quantitative SGA Screens

Yeast Genetic Interaction Network Global Level DNA replication

Yeast Genetic Interaction Network Global Level Vesicle- mediated transport DNA replication

Ribosome, Translation Mitochondria Vesicle- mediated transport Glycosylation & cell wall Polarity & cell morphogenesis DNA replication and repair Chromosome segregation and mitosis Nuclear-cytoplasmic transport Chromatin & transcription Peroxisomes Amino acid biosynthesis RNA processing Nuclear migration Protein Degradation Yeast Genetic Interaction Network Global Level

Can we recapitulate synthetic lethality in mammalian cells?

yfg1 yfg2 Dead yfg2 yfg1 yfg2 Normal Tumor yfg1 yfg2 SL interactions identified in yeast could be investigated as a candidate for novel therapeutic target

Potential therapeutic targets

Synthetic lethality in model organisms and human cancers

RAD54B depletion underlies CIN

FEN1 down-regulation underlies synthetic lethality in RAD54Bdeficient human cells

Blue circle, SiRNA targeting central gene alone Red circle, SiRNA targeting cancer gene alone Yellow triangles, predicted viability of double siRNA treatment Green circles, observed viability of double siRNA treatment

Solid grey line, interaction observed in both S. cerevisiae and HCT116 cells. Green dashed line, interaction observed only in S. cerevisiae. Orange dotted line, interaction observed only in HCT116 cells. Mammalian genetic interaction network

Screening for FEN1 inhibitors