Mutations (Lecture 1-5)

What is the classical genetics’ definition of a mutation?

a heritable change in a gene resulting in a change in phenotype

What is the modern molecular term for a mutation?

a heritable change to the sequencee of a DNA molecule

What did George Beadle and Edward Tatum discover?

that genes work by making enzymes

What hypothesis is associated with Beadle and Tatum?

1 gene 1 enzyme

What organism was used in the Beadle and Tatum experiment?

neurospora — a common eukaryotic bread mold (fungus)

What kind of auxotrophs were used in the Tatum and Beadle experiment?

arginine auxotrophs

What is an auxotroph?

a microorganism that can only grow in the presence of specific growth supplements (usually an amino acid)

What 3 genes were focused on in the Beadle and Tatum experiment?

genes that caused the arginine auxotrophy — arg-1, arg-2, and arg-3. These are located on 3 different chromosomes

What were the 4 different conditions used for growing in the Tatum and Beadle experiment?

-Minimal Media

-Minimal Media + ornithine

-Minimal Media + citruline

-Minimal Media + arginine

What were the growth results of the Tatum and Beadle experiments for each condition?

Wild type mold: grew in all 4 growth conditions

Mutant in arg-1: did not grow in Minimal Media, but grew in other 3 conditions

Mutant in arg-2: did not grow in Minimal Media or MM + ornithine, but grew in other 2 conditions

Mutant in arg-3: only grew in Minimal Media + arginine

Pathway of gene products based on results of Tatum and Beadle experiment

-arg-1 makes enzyme 1

-Enzyme 1 catalyzes the production of ornithine

-arg-2 makes enzyme 2

-Enzyme 2 catalyzes the synthesis of citrulline from ornithine

-arg-3 makes enzyme 3

-Enzyme 3 catalyzes the synthesis of arginine from citrulline

Conclusions from Tatum and Beadle experiment

-3 classes of mutants: arg1, arg2, and arg3

-each gene gives rise to a different enzyme that catalyzes a different chemical reaction

-Basis of one-gene-one-enzyme hypothesis

-Still did not show how genes and enzymes are related

Why should we care about mutations?

-they cause genetic diversity, and are the basis of evolution

-they provide insight into how genes and their products work

-they can lead to cancer and diseases

-they can provide antibiotic resistance in microbes

-they can improve commercial properties of animals, plants, and microbes (GMOs)

Why should we study the genetics of microbes?

-we can perform genetic screens that are not possible in higher eukaryotes

-the mechanisms and nature of mutations are effectively the same in microorganisms as in eukaryotes

What bacteriophage did Salvador Luria and Max Delbruck analyse?

T1; infects and kills E. Coli

General Process of Lytic Bacteriophage Lifestyle

-phage adsorbed by host cell

-phage’s nucleic acid is read by the host

-phage proteins and materials are synthesized by the host

-Host chromosome is degraded

-Phages assemble and the host cell lyses

-Phages are released

Luria and Delbruck Experiment

-Infected lots of E. Coli with T1 bacteriophage

-Most of the E. Coli dies, BUT some were resistant

-The resistant E. Coli survived and gave colonies

-The progeny of the survivors were also resistant to T1

Conclusion of original Luria and Delbruck Experiment

resistance is an inheritable genetic trait

What were the 2 hypotheses after the Luria and Delbruck experiment for how the mutant bacteria arose?

1) Induced mutation (adaptive response): the presence of the phage induced some bacteria to become resistant

2) Darwinian evolution: resistant mutations arose spontaneously in the population and were then selected for by phage addition

Why did Luria and Delbruck develop the fluctuation test?

to determine how the phage-resistant mutation arose

Principle / General Ideas of Fluctuation Test

-If mutations are induced by the phage, resistant cells will not appear in the lineage before the phage is added

-If mutations are spontaneous and simply selected for by the phage, then the mutations must be present in cell generations before the phage is added

-So, if the mutations are induced, each “batch” should have roughly the same number of resistant cells

-BUT, if the mutation is spontaneous, then different batches will have different numbers of resistant bacteria present

What are the steps of the fluctuation test?

1. expose different cultures / batches of E. Coli bacteria to the T1 bacteriophage

2. spread cultures onto agar plates to let the survivors grow

3. count the colonies of survivors for each culture

How to perform fluctuation test experimentally

take one large (bulk) culture, split it into small portions, and analyze the number of resistant bacteria per portion

Why is it called the “fluctuation” test?

if the mutation is spontaneous, you will see a wide “fluctuation” in the number of resistance colonies when you divide the culture into small batches

Results from Luria and Delbruck’s fluctuation test

-wide fluctuations in the number of resistance colonies

-consistent with mutations arising spontaneously and mutant bacteria were simply selected for by T1 phage

Limitations of Fluctuation Test Experiment

-bacteria were rapidly killed (did not have time to adapt)

-it can take several generations for resistance mutations to give rise the resistance phenotype

-still allowed us to know that mutations are pre-existing and are selected for

Goal of Ames Test

detection of mutagens

What is a mutagen?

an agent capable of increasing the mutation rate in organisms

-Can be radiation or chemicals

Examples of mutagens

UV, X-rays, carcinogens, processed foods, cosmetics, cleaning products

Why do we use micro-organisms to identify mutagens in the environment?

-easy to screen in large numbers

-more sensitive screening

-genetic pathways are better understood

-more ethical than using eukaryotic organisms

Who developed the Ames test?

Bruce Ames

Principle of original Ames test

-Salmonells strains that are His-auxotrophs are spread onto a growth medium without Histidine present

-Any bacteria that grow have had a 2nd mutation (in the His gene) and are called “revertants”

-If the test compound (potential mutagens) increases the number of revertants, it is likely a mutagen

Revertant

an auxotrophic bacterial cell that has had a second mutation, allowing it to grow on media lacking the amino acid it is auxotrophic for

Example of Ames test experiment with Aflatoxin

-Aflatoxin is the test compound (potential mutagen)

-His- strains of auxotrophic salmonella were exposed to different concentrations of Aflatoxin

-Conclude that Aflatoxin DOES increase the frequency of mutations, and is a mutagen

Why did Ames’s team develop strains of Salmonella with different kinds of mutations in the His genes?

-different mutagens cause different kinds of mutations

-each strain needs a different kind of mutation (induced by a different mutagen) to cause reversion back to the wild type

What was a major limitation to the Ames test? What was the solution?

Problem: many chemicals that are not mutagenic to bacteria are mutagenic to mammals because they are converted to an active form by enzymes in the liver

Solution: prepare liver extracts and incorporate them into the medium used in the Ames test so that the active form of the mutagens are present

What are some ways the Ames test has been improved?

-incorporate liver enzymes

-use bacterial strains with mutations in the cell envelope (rfa genes) to make them more permeable to chemicals

-use strains with a defective protein for DNA repair (uvrB gene)

-incorporate a plasmid that enhances the effectiveness of some mutagens (pKM101)

What is the overall purpose of all of the improvements to the Ames test?

to increase effectiveness of mutagens (have MORE mutations in the bacteria)

Point mutations

occur at a single point (usually a single base pair) in the genome; also known as an SNP (single-nucleotide polymorphism)

Types of SNPs

-substitution

-indels

Substitution Mutations

one base pair is swapped for another

Indel Mutations

a base pair is inserted or deleted from the genome

What types of mutations are NOT point mutations?

deletion (loss) or large-scale rearrangements of DNA or insertion of mobile genetic elements

Can point mutations be spontaneous?

yes, many have no evidence of external causes

What process to point mutations need to become permanent?

need DNA synthesis to become permanently encoded in the genome

What are some ways that point mutations can arise?

-mistakes in DNA Polymerase proofreading

-tautomerization of bases

How can tautomerization of bases lead to point mutations?

-sometimes, a base transiently flips into a different isomer with different base-pairing properties

-Cytosine can tautomerize into its rare form, where it pairs with A instead of G

-Then, because it is paired incorrectly, one of the daughter strands during DNA replication will permanently have a mutation in that spot (an AT where it should have been a GC)

DNA Proofreading Mechanism

-DNA Polymerase “checks” each new base pair

-If there is a mistake, the enzyme will back up and remove the incorrect base pair

-This is 99% effective

-After correcting the mistake, the DNA synthesis returns

Which two enzymes are important as DNA proofreaders?

DNA Polymerase I and DNA Polymerase III

-they excise the mismatched base and can then add the correct complementary base

What happened to mice without proofreading mechanisms?

-increased mutation in mitochondrial DNA

-premature ageing

Mismatch Repair System (MMR)

-second mechanism for reducing spontaneous mutations

-can detect non-matched “pairs” of bases in DNA (mismatches)

-determines which of the bases is wrong

-removes the wrong base

Process / Steps of MMR

1. detect mismatches in newly replicated DNA with the MutS protein

2. Bind MutS to distort the DNA double helix; this distortion recruits MutL and MutH

3. MutH cuts the “new” strand of DNA containing the wrong base

How does MMR know which strand is “new” and which is parental?

-Adenine bases are methylated in DNA after the strand is formed

-So, parental strands will be methylated while new strands will not yet be methylated

-Now, MMR can distinguish between parental and new strands and determine which of the bases is “wrong”

On which atom is Adenine methylated?

N6

MutH

-recognizes new strand by adenine methylation at GATC sequences

-endonuclease activity cuts unmethylated strand

MutS-MutL

-also activates excision

-involved DNA helicase (UvrD) and exonucleases

Role of DNA Pol III in MMR

repairs the ss-gap

Role of DNA ligase in MMR

generates continuous covalent DNA backbone

Deamination

-type of mismatch that removes an amine, changing C to U

-U pairs with T, so a CG base pair is turned into an AT base pair

When do indel mutations occur?

during DNA replication

Streisinger Model

-DNA “slippage” leads to loss or incorporation of new base pairs into DNA

-this model fits the experimental data

-Loops in single-stranded regions of DNA are stabilized by “slipped mispairing” of repeated sequences during replication

Ways to prevent spontaneous mutations

-Inherent accuracy of DNA Polymerase (mistakes are 1 in 100,000)

-Proofreading mechanism (corrects 99% of mistakes)

-MMR System (corrects 99% of the remaining mistakes after proofreading)

What is the overall spontaneous mistake rate in DNA?

1 per 1 billion base pairs

What causes tuberculosis?

infection with Mtb bacteria

What is the treatment regimen for someone with TB?

must take lots of pills + an injection daily

BPaL

-new drugs to target resistant Mtb

-Bedaquiline, pretomanid, linezolid

What is the main problem with treating TB?

drug resistance

miniON

a small device used for sequencing with the goal to target TB drug resistance and provide better diagnostics

Examples of Mutagens

-EMS (ethylmethylsulphonate)

-AP (aminopurine)

-Aflatoxin

-UV light

What did Jeffrey Miller discover?

he used the lacI gene of E Coli as a model system to learn that different mutagens cause different mutations in DNA

How do chemical mutagens work?

-they modify base pairing properties

-DNA Polymerase then incorporates the wrong base during replication

How does EMS work?

-changes the G base to O-6-ethylguanine by adding an O-ethyl to C6

-this creates an ester

-O-6-ethylguanine then pairs with T instead of C

-works as an alkylating agent (changes GC to AT mutations)

What is an alkylating agent?

a mutagen that causes GC —> AT mutations

How does 2-AP (2-aminopurine) work?

acts as a base analogue by replacing adenine

-often, it pairs with T (which is good), but it can sometimes also pair with C (bad)

-If it pairs with C during replication, an AT base pair will change to a AC and then GC

How does Aflatoxin work?

-produced by fungi

-chemically reacts with G bases in DNA, causing GC to TA mutations

-Generates apurinic sites

What is an apurinic site?

-depurination occurs when a purine base (A or G) is lost from the DNA

-this creates an apurinic site, or a site without a purine

-During DNA replication, there is a “blank” where the purine should be

-A base (usually Adenine) can be inserted opposite the blank

-This changes the sequence of base pairs

How does “bypass” DNA Polymerase contribute to making mutations?

-most DNA is made from DNA Polymerase with proofreading activity (like DNA Pol I and III), and these enzymes cannot synthesize past “damaged” DNA lesions (like apurinic sites)

-Instead, you need to use a special “bypass” DNA Polymerase to do this process, known as Translesion DNA Synthesis

What is the problem with Bypass DNA Polymerase?

it is much more likely to incorporate wrong bases even when the DNA is not damaged (compared to normal DNA Polymerases), leading to more mutations

Steps of Trans-Lesion DNA Synthesis (TLS)

-A stalled DNA Polymerase initiates the process by triggering recruitment of TLS Polymerase

-TLS Polymerase synthesizes past the lesion

-Once extension of DNA passes the lesion, the TLS polymerase is replaced by replicative DNA Polymerase

Do humans and E. Coli have different TLS mechanisms?

No, it is conserved

How typical are the different types of mutagens?

-Many chemical mutagens alter bases and affect base pairing (like EMS)

-Some mimic normal bases and are incorporated into DNA, then later pair with the wrong base (like 2-AP)

-Some result in absence of a pairable base during synthesis (like aflatoxin)

What process do all mutagen mechanisms require to become mutations?

DNA replication

What is a pre-mutagenic lesion?

a change to DNA that may lead to a mutation

What is needed to get from a pre-mutagenic lesion to a mutation?

at least 1 round of replication

When do DNA repair systems repair “damaged” DNA?

before replication

EMS Damage Repair

alkyltransferases are repair proteins that transfer the alkyl (ethyl) group from the DNA base to the protein

Repair of Apurinic Sites

-AP Endonuclease recognizes apurinic sites and cuts the DNA strand

-Defective DNA and some nearby DNA is then removed by a set of excision exonucleases

-DNA synthesis fills in the gap

-This is an example of excision repair

How does UV radiation cause mutations?

leads to the creation of photodimers

What is a photodimer?

when UV radiation causes adjacent T bases to become covalently crosslinked

How do photodimers lead to mutations?

-they fail to base pair correctly during replication

-even though translesion DNA Polymerase can replicate past them, it may incorporate the wrong base

What are the 2 mechanisms for repairing photodimers?

1) Nucleotide excision repair

2) Photolyase enzyme

Nucleotide excision repair

similar to the repair of apurinic sites, works to repair photodimers

Photolyase enzyme

uses energy from white light to convert photodimers back to pyrimidines (photorepair)

Do the molecular mechanisms identified in bacteria also apply to eukaryotes?

Yes!

-molecular nature of mutations is the same in humans and bacteria

-higher eukaryotes have DNA repair systems similar to those in bacteria

-Defects in DNA repair are associated with neurodegenerative disorders and xeroderma pigmentosum

What is xeroderma pigmentosum?

sensitivity to sunlight

Colibactin

-molecule secreted from some E. Coli that can live in the human gut

-can react with DNA in human cells and cause mutations (mutagen)

-related to colorectal cancer

What was the large concept discovered by Seymour Benzer?

complementation

What experimental system did Benzer use in his complementation work?

bacteriophage T4

Why did Benzer use a bacteriophage as his experimental system?

-easily and rapidly grown

-genetically simple

-must have similar genetic mechanisms to the host cells and can thus act as “probes” of genetic mechanisms

-easy to analyze billions at a time

What are bacteriophage plaques?

“holes” in a lawn of bacteria where bacteria have been killed by the phage (clear areas on an agar plate)