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.

http://www.biomedcentral.com/1471-2156/3/4/figure/F5

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 analogsBase modifying agentsIntercalators Other classes

Millions of natural and synthetic compounds

2-AP: 2- aminopurine

Analog of adenine that can pairwith cytosine in its protonated state

Normal pairing14

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 DNAas a base .

The ionized form base pairs with guanine.

Normal pairing

Transition

Rare form of BrU in Template

Mutagens מוטגנים

Chemical Agents

Base analogsBase modifying agentsIntercalators

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

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 induceGC to TA transversions

Usually an A is inserted instead of the depurinated site

2-APEMS

NG

Mutation rate, a question of balance

Mutations DNA repair

Mutagens

Chemical Agents

Base analogsBase modifying agentsIntercalators

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 intercalatedposition, an agent can cause single-nucleotide-pair insertions or deletions

Intercalators

DAPI hCia2-GFP α-Tub1 MergeM

etap

has

eT

elop

has

eC

ytok

ines

is

TelophaseAnaphaseMetaphaseCIA knock-downControl

TelophaseAnaphaseMetaphase

DA

PI

α-T

ub

1M

erge

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 Transition

Transversion

Transversion

Transversion

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 testA 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 chemicalcompounds.

The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound

Bruce Ames1928-today

University of California,Berkely

TA100- sensitive for reversion by base pair substitutionTA 1535/8 frameshift

Ames test

Strains inactive forBER and prone forEntry 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 functions transcription mechanisms**cell cycle** transcriptional regulationorganelle biogenesis chromatin modificationsecretion* signal transductionprotein targeting mechanisms protein degradation*chromosome replication DNA repairgenome dynamics retroviral packagingprions recombination mechanismsageing function of new genesmetabolism protein modification

*Lasker Award **Nobel Prize

"for their discoveries of key regulators of the cell cycle"

Lee HartwaellPaul Nurse Tim 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. Rothman Thomas C. SüdhofRandy W. Schekman

The Nobel Prize in Physiology or Medicine 2013

40

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 G1

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

DELETION LIBRARYDELETION LIBRARY

• Availability:Availability: available commercially available commercially

• Description:Description: collection of strains deleted in all the non-essential genes collection of strains deleted in all the non-essential genes..

• Creation:Creation: replacing the gene in question by barcod containing selectable replacing the gene in question by barcod containing selectable marker based on homologus recombinationmarker based on homologus recombination

gene1::KmX

KanMX4

GENE1

gene1::KmX gene2::KmX gene3::KmX gene4::KmX gene4700::KmX

KmX is a gene that provides resistance to the drug G418

UptagU1 U2 D2D1 Downtagxxx::kanMX4

yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX

2 main ways of studying the knockout collection:

.1Plate-based

.2Chip-based

Serial analysis of deletion strains(plate based)

Apply Selection

Identify deletion strains with growth defects

1

2

3

6,000

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

Large-scale Mapping of Genetic and Interactions in Yeast

Synthetic Lethal Genetic Interaction in Yeast

Synthetic Lethality

yfg1

yfg2

Dead

Viable Viable

yfg1

yfg2

Synthetic LethalityA

B

a

B

XA

b

Viable Lethal

a

b

Wild-type Viable

XXX

Functional Relationships

Essential biological functionSL interaction

Pathway B

A2

A3

B1

B2

B3

Pathway A

A1A1

A2

A3B1

B2

B3

Complex A Complex B

How can we detect synthetic lethal interactions between a mutant ofInterest (query gene)

and other genes

B Mating

Heterozygous diploid

Tetrad

BABAbaba

BaBabAbA

BABababA

NPD PDT TT

Tetrad Dissection

Meiosis

a b AB 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)

BABAbaba

BaBabAbA

BABababA

NPD PDT TTTetrad

Tetrad Dissection

bni1∆ bnr1∆

We ask which mutation is synthetically lethal with our query mutant

yfg1::ClonNAT

Mating

yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX

Query gene

Meiosis

4700 heterozygous diploids

Tetrads

The problem is that it is impossible to perform 4700 tetrad dissections

wt

wt wt wt wt wt

NPDPDTTT

a b

A B

a b

A B

MATa

MATα MATα

MATaa

bA

BMATa MATα

MATαMATa

a B

bA

a BMATa MATα

MATαMATabA

A B

a b

yfg1::ClonNAT

yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX

Query gene

Mating

4700 heterozygous diploidsMeiosis

Tetrads

We ask which mutations are synthetically lethal with our query mutantThe 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::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX

yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT

Use the haploid selection marker for selection

Multiplicative Model Expected Double Mutant Fitness

wt 1

0.5

0.5

Fitness

“Neutral” Expected Result, Multiplicative Model (additive)

0.25

a

b

ab

R. Mani F. Roth et. al., PNAS 2008 Mar 4;105(9):3461-6. Epub 2008 Feb 27

D. Segre, R. Kishony et al., Nature Genetics 37, 77 - 83 (2004)

S. R. Collins, N. Krogan, J. Weissman, Genome Biol 2006;7:R63. “S-Score””

Two Basic Types of Genetic Interactions

wt 1

0.5

0.5

“Negative” Synthetic Lethal

“ Neutral”Expected Result

a

b

ab

Fitness (colony size)

1

Two Basic Types of Genetic Interactions

wt 1

0.5

0.5

“ Neutral”Expected Result

0.5

“Positive”e.g. two genes whose products are inthe same nonessential complex/pathwayEpistasis

a

b

ab

FitnessFitness (colony size)

0.5

0.5

0.5

Proteincomplex

A

B C

Non-functionalB C

Non-functionalA

C

Non-functionalC

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 Interactions204 Genes

8 SGA Screens:291 Interactions204 Genes

Genetic Interaction Network132 Screens

4000 Interactions1000 Genes

~200,000 Interactions/genome

Amy Tong, Fritz Roth et al., Science 303:808-813 (2004)

Tong et al., 2004. Science 303: 808 - 813

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

dyn

ein

SGA analysis clusters related genes

Array gene clusters

Qu

ery

gen

e cl

ust

ers

~2000 Quantitative SGA Screens

Yeast Genetic Interaction NetworkGlobal Level

DNA replication

Yeast Genetic Interaction NetworkGlobal 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 migrationProtein

Degradation

Yeast Genetic Interaction NetworkGlobal Level

Can we recapitulate synthetic lethality in mammalian cells?

yfg1

yfg2

Dead

yfg2yfg1

yfg2

yfg2

Normal

Tumoryfg1

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 bothS. 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