DNA damage and Mutagenesis

Question 1

What can mutations be?

Answer 1

Spontaneous or induced

Question 2

Examples of spontaneous mutations - consequences of abnormalities in biological processes

Answer 2

-errors by DNA polymerase in replication
-metabolic processes leading to free radical production e.g superoxides
-spontaneous changes in base structures giving altered base pairing

Question 3

What are induced mutations?

Answer 3

-Environmental agents that enter cells and alter DNA
-Occur more frequently than spontaneous mutations
-Agents involved called mutagens - chemical or physicak

Question 4

What are base analogues and what is an example

Answer 4

Mutagens that have structures like normal bases in DNA
-Once incorporated, they have diff base pairing properties than the bases they replace

e.g 5-bromouracil is an analogue of thymine when in its keto form but then behaves like cytosine in its enol form and bonds with guanine
-sometimes behaves in keto form and sometimes in enol form

e.g 2 aminopurine is an adenine analogue that pairs w thymine but can behave like guanine and pair w cytosine

Question 5

Write out 5-bromouracil 3 cycles on replication

Question 6

What do base modifications do?

Answer 6

Alters bases already in DNA sequences e.g deaminating agents, alkylating agents

Question 7

What do deaminating agents do?

Answer 7

Nitrous acid deaminates cytosine to produce uracil which base pairs with adenine and causes GC to AT transitions upon subsequent replication

Deamination of adenine to guanine analogue hypoxanthine results in AT to GC.

Question 8

What do alkylating agents do and what are examples of this?

Answer 8

-Add methy and ethyl groups to bases and alter base pairing
-e.g nitrogen mustards and ethyl methansulfonate (EMS)

EMS changes guanine into O6-ethylguanine which binds to thymine so GC to AT

Question 9

What are intercalating agents?

Answer 9

-Flat molecules that slip in between adjacent base pairs and interfere w DNA replication e.g ethidium bromide
-leads to insertion mutations that cause frameshifts because extra nucleotide has been added

Question 10

What does UV radiation cause?

Answer 10

-Crosslinks adjacent pyrimdines on the same strand forming pyrimidine dimers - interferes w replication
-Usually Ts
Other radiation types like xrays and gama cause single and ds DNA breaks

Question 11

What ways can DNA damage repair be done?

Answer 11

1. directly undo damage - photoreactivation or O6 methylguanine methyl transferase
2. remove damaged section and fill w new undamaged DNA

Question 12

What happens in direct reversion - photoreactivation

Answer 12

-Found in many organisms but not humans
-Repair of damage caused by UV light
-Enzyme is photoreactivating enzyme or DNA photolyase

Question 13

What is the mechanism of photoreactivation?

Answer 13

1. Enzyme detects and binds the damaged DNA (dimer)
2. Enz absorbs light which activates it
3. Breaks bond holding dimer together
4. Enzynme dissociates and damage is repaired

(detects as dimer DNA not exact same- slight kink so breaks bond between both thymines)

Question 14

What happens in O6 methylguanine methyl transferase?

Answer 14

O6 alkylations on guanine residues (guanine that has been methylated by alkylating agents) can be directly reversed by the ‘suicide’ enzyme (not regenerated) which accepts the alkyl group onto one of the cysteine amino groups

-Inactivates enzyme but restores guanine
-The enzyme in E.coli is induced by DNA alkylation (trigger) - cells previously exposed are more resistant to alkylating agents

Question 15

What are the types of excision repair (ER) - removal and replacement of damage?

Answer 15

-Base ER - small, common changes to DNA
-Nucleotide ER - more drastic changes to bases that often distort helix
-Mismatcg excision

Question 16

What is the mechanism of base excision repair?

Answer 16

-A damaged base (alkylated/deaminated) is recognised by a DNA glycosylase and brings about a distortion in DNA that leads to breaking of the glycosidic bond between base and sugar - leaving an apurinic or apyrimidinic site (AP)
-This AP site is recognised by AP endonuclease that cuts the strand at the 5’ side of the site
-In e-coli DNA phosphodiesterase removes the remaining sugar-phosphate.
-DNA polymerase I performs a repair synthesis by degrading DNA in the 5’ to 3’ direction and filling with new DNA
-DNA ligase seals the repaired nick

Question 17

What fills the missing nucleotides in euks?

Answer 17

DNA polymerase ß fills in the missing nucleotides but as it cant proofread, AP endonuclase performs this function

Question 18

What happens in nucleotide excision repair?

Answer 18

-more general mechanism for removing damage that causes distortions in DNA

Mechanism:
-A repair endonuclease (uvrABC) removes damaged bases (dimers) along with bases on either side of the damage
-The gap (approx 12 nts) is filled by DNA pol1 and sealed by DNA ligase

Question 19

What happens in mismatch excision repair?

Answer 19

-Repairs errors that occur during DNA synthesis
-Mismatched nucleotides are excised and replaced with the correct nucleotide
-Changes being detected are subtle and is generally a substitution with what is a normal base in DNA
-MutS is the protein that identifies this errors and needs to identify which base in the mismatch is incorrect- which strand was synthesised most recently
-Methylation of sequences is used to discriminate parental from daughter strands

-parental dna will be methylated as DNA becomes methgylated when has been around for awhile

Question 20

Methylation of sequences in e-coli

Answer 20

In E-coli, GATC sequence motifs are methylated shortly after replication
-The enzyme complex MutH-MutL-MutS or MutHLS catalyses mismatch repair in E.coli

Question 21

What are 2 other repair systems?

Answer 21

Recombination repair

Induced repair system (SOS system)
-LexA protein represses many of the genes involved in DNA replication and repair (low levels)
-When extensive damage occurs to DNA recA brings about proteolytic digestion of lexA
-Many more copies of these enzymes are produced, accelerating replication and repair
-However, the system is error prone and itself introduces mutations into DNA as it repairs as working so rapidly but sometimes these are easy fixed