Lecture 4 Flashcards
What is a mutagen
an agent capable of increasing the frequency of mutaion
What kind of mutaions do mutagens mainly induce?
- most increase the frequency of substitution mutations
- some result in indels
3 examples of mutagens
- EMS
- aminopurine
- UV light
Different mutagens have different ____ - they can use different ____ in the ____
Effects
Mutation
DNA
How do chemical mutagens work?
- by modifying base - pairing properties
- DNA polymerase then incorporates wrong bases during DNA replication
3 key mutagens
What is EMS and what mutaion does it cause
- EMS is an alkylating agent
- causes GC —-> AT mutations
(Gaunine changes chemical properties which makes binding to thymine more favourable)
EMS pre mutaion lesion
- ethyl on guanine
EMS mutaion diagram
2 amino purring mutaion - a base analogue - what happens when adenine changes to 2-AP?
- most often pairs with thymine but can also base - pair with cytosine
- if this happens during replication, will lead to an A-T basepair being changed to A-C and subsequently G-C
What is aflatoxin produced by?
Fungi
What does aflatoxin do?
- chemically reacts with guanine (G) bases in DNA (interferes with back bone)
- causes GC —> TA mutations
Reaction of aflatoxin B with DNA generates _________ _____ this can lead to _______
Apurinic sites
Mutation
What are apurinic sites?
Apurinic sits are the result of depurination - a purine base (adenine or guanine) is lost from the DNA
- Apurinic site thus is a sit without a purine
Why do apurinic sites cause mutations?
- during DNA replication, there is a blank where the purine should be
- a base (often an adenine) may be inserted opposite the blank
- this can change the sequence of base-pairing
Can DNA polymerases synthesise past “damaged” DNA (lesions) such as apurinic sites? What is used instead?
No
Instead special “bypass” DNA polymerases do this (translesion DNA synthesis)
Are bypass DNA polymerases as accurate as normal DNA polymerase? What does this result in?
No, they are much more likely to incorporate the wrong base even when the DNA is not damages leading to mutaions
Process of trans-lesion DNA synthesis
It is initiated by stalled DNA polymerase (stalled cos there is a lesion and it doesn’t know what to do), which triggers recruitment of a TLS polymerase that synthesises past the lesion
Once extension passes the lesion, the TLS polymerase is replaced by the replicative DNA polymerase
3 main effects of mutagens causing mutaions and what they all require
- chemical mutagens alter bases in the DNA and affect base-pairing (like EMS)
- some mutagens “mimic” normal bases and become incorporated into the DNA, then later pair with the “wrong” bases (like 2-AP)
- other mutagens result in the absence of a “pairable” base during DNA synthesis (like aflatoxin)
ALL mechanisms require DNA replication to become mutations!!!! <—- take home message!
What is a pre-mutagenic lesion?
A change to the DNA that may lead to a mutation
Where does DNA repair systems take place? And why?
- at least one round of DNA replication is needed to get from a pre-mutagenic lesion to a mutation
- DNA repair takes place at the pre-mutagenic lesion (because ones the mutation is “established”, it is too late)
- DNA repair systems repair “damages” DNA BEFORE it is replicated
How do you repair EMS damage?
- repair the proteins using alkyltransferases
- the (alkyl) ethyl group is transferred from the base in the DNA to the protein
- thus it can still pair with its correct pair
Process of Repair of Apurinic sites
- an enzyme (AP endonuclease) recognises an apurinic site and cuts the strand of DNA that contains it
- the defective DNA and some adjacent DNA is then removed by a set of enzymes (excision exonucleases
- the cap is filled my DNA synthesis
- an example of excision repair
How radiation (e.g ultraviolet light) causes mutations
- adjacent thymidine (T) bases in the DNA can become covalently cross-linked - photodimers
- these fail to base-pair properly during DNA replication
- Translesion DNA polymerases can replicate past these but may incorporate the wrong base
How to repair photodimers
- Nucleotide excision repair
- similar to repair apurinic sites - Photolyase enzyme
- this uses energy from white light to convert photodimers back to pyrimidines (photorepair)
Do the molecular mechanisms (repair systems) in bacteria also apply to eukaryotes ?
- DNA sequencing shows that the molecular nature of mutations in humans is the same as in bacteria
- higher eukaryotes have repair systems that are equivalent to those that have been identified in bacteria
- defects in DNA repair have been associated with neurodegeneratice disorders and xeroderma pigmentosum - exceptional sensitivity to sunlight
How some e.coli bacteria can use mutagens as weapons
- some coli bacteria that can live in the human gut secrete a compound called Colin actin
- this can react with DNA in human cells and lead to mutations
- related to colorectal cancer
Take home messages
Researcher that used experimental system bacteriophage T4
Seymour benzer
What did Seymour benzer do?
Experimental system bacteriophage T4
- dont mix up with T1 from earlier lecture
What is a bacteriophage
Viruses that infect bacteria
Why did benzer use bacteriophage ?
- easily and rapidly grown
- genetically amongst the simplest organisms
- they have similar genetic mechanisms to the host cells
- can analyse many billions very easily
What are bacteriophage plaques ?
“Holes” in a lawn of bacteria where bacteria have been killed by the phage
rII gene mutant phage vs wild-type bacteriophage T4
- Wild-type T4 can infect two different kinds of E.coli bacteria, strains K and B
- rII mutant phage forms large plaques on E.coli strain B but CONNOT form plaques on E.coli strain K
Definition of complementaion
Production of wild-type phenotype when two mutant haploid genomes bearing different recessive mutations are present in the same cell
What did benzer do to show compliemntaiton
- infected pairs of rII mutants into E.coli K12
Benzer complementaiton system diagram
