Lecture 4

Question 1

What is a mutagen

Answer 1

an agent capable of increasing the frequency of mutaion

Question 2

What kind of mutaions do mutagens mainly induce?

Answer 2

• most increase the frequency of substitution mutations
• some result in indels

Question 3

3 examples of mutagens

Answer 3

• EMS
• aminopurine
• UV light

Question 4

Question 5

Different mutagens have different ____ - they can use different ____ in the ____

Answer 5

Effects
Mutation
DNA

Question 6

How do chemical mutagens work?

Answer 6

• by modifying base - pairing properties
• DNA polymerase then incorporates wrong bases during DNA replication

Question 7

3 key mutagens

Answer 7

Question 8

What is EMS and what mutaion does it cause

Answer 8

• EMS is an alkylating agent
• causes GC —-> AT mutations

(Gaunine changes chemical properties which makes binding to thymine more favourable)

Question 9

EMS pre mutaion lesion

Answer 9

• ethyl on guanine

Question 10

EMS mutaion diagram

Answer 10

Question 11

2 amino purring mutaion - a base analogue - what happens when adenine changes to 2-AP?

Answer 11

• 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

Question 12

What is aflatoxin produced by?

Answer 12

Fungi

Question 13

What does aflatoxin do?

Answer 13

• chemically reacts with guanine (G) bases in DNA (interferes with back bone)
• causes GC —> TA mutations

Question 14

Reaction of aflatoxin B with DNA generates _________ _____ this can lead to _______

Answer 14

Apurinic sites
Mutation

Question 15

What are apurinic sites?

Answer 15

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

Question 16

Why do apurinic sites cause mutations?

Answer 16

• 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

Question 17

Can DNA polymerases synthesise past “damaged” DNA (lesions) such as apurinic sites? What is used instead?

Answer 17

No
Instead special “bypass” DNA polymerases do this (translesion DNA synthesis)

Question 18

Are bypass DNA polymerases as accurate as normal DNA polymerase? What does this result in?

Answer 18

No, they are much more likely to incorporate the wrong base even when the DNA is not damages leading to mutaions

Question 19

Process of trans-lesion DNA synthesis

Answer 19

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

Question 20

3 main effects of mutagens causing mutaions and what they all require

Answer 20

• 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!

Question 21

What is a pre-mutagenic lesion?

Answer 21

A change to the DNA that may lead to a mutation

Question 22

Where does DNA repair systems take place? And why?

Answer 22

• 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

Question 23

How do you repair EMS damage?

Answer 23

• 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

Question 24

Process of Repair of Apurinic sites

Answer 24

• 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

Question 25

How radiation (e.g ultraviolet light) causes mutations

Answer 25

- 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

Question 26

How to repair photodimers

Answer 26

1. Nucleotide excision repair - similar to repair apurinic sites 2. Photolyase enzyme - this uses energy from white light to convert photodimers back to pyrimidines (photorepair)

Question 27

Do the molecular mechanisms (repair systems) in bacteria also apply to eukaryotes ?

Answer 27

- 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

Question 28

How some e.coli bacteria can use mutagens as weapons

Answer 28

- 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

Question 29

Take home messages

Answer 29

Question 30

Researcher that used experimental system bacteriophage T4

Answer 30

Seymour benzer

Question 31

What did Seymour benzer do?

Answer 31

Experimental system bacteriophage T4 - dont mix up with T1 from earlier lecture

Question 32

What is a bacteriophage

Answer 32

Viruses that infect bacteria

Question 33

Why did benzer use bacteriophage ?

Answer 33

- easily and rapidly grown - genetically amongst the simplest organisms - they have similar genetic mechanisms to the host cells - can analyse many billions very easily

Question 34

What are bacteriophage plaques ?

Answer 34

“Holes” in a lawn of bacteria where bacteria have been killed by the phage

Question 35

rII gene mutant phage vs wild-type bacteriophage T4

Answer 35

- 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

Question 36

Definition of complementaion

Answer 36

Production of wild-type phenotype when two mutant haploid genomes bearing different recessive mutations are present in the same cell

Question 37

What did benzer do to show compliemntaiton

Answer 37

- infected pairs of rII mutants into E.coli K12

Question 38

Benzer complementaiton system diagram