Module 8 - Causes of DNA mutation and repair

Question 1

Mutation

Answer 1

An alteration in the nucleotide sequence of the DNA

Question 2

Two types of mutation

Answer 2

Spontaneous mutations - occur during DNA replication due to base tautomerism

Mutagens - chemical or environmental agents causing changes in DNA through base analogs or direct structure changes

Question 3

Tautomers: what are they, what are the examples, and what effect does this have on daughter DNA molecules?

Answer 3

Isomers of bases with slightly different structures, usually due to the movement of a double bond and one hydrogen

• Amino-adenine bonds with thymine, imine-adenine bonds with cytosine
• Keto-thymine bonds with adenine, enol-thymine binds with guanine
• Keto-guanine bonds with cytosine, enol-guanine bonds with thymine
• Amino-cytosine bonds with guanine, imino-cytosine bonds with guanine

One out of four granddaughter DNA molecules will be a mutated DNA molecule

Question 4

Difference between amino-adenine and imino-adenine?

Answer 4

Amino-adenine (R-(H₂N\C=N)-R) bonds with thymine, imine-adenine (R-(HN\C-NH)-R) bonds with cytosine

Question 5

Difference between keto-thymine and enol-thymine?

Answer 5

Keto-thymine (R-(N=C/OH)-R) bonds with adenine, enol-thymine (R-(HN-C//O)-R) binds with guanine

Question 6

Difference between keto-guanine and enol-guanine?

Answer 6

Keto-guanine (R-(N=C/OH)-R) bonds with cytosine, enol-guanine (R-(HN-C//O)-R) bonds with thymine

Question 7

Difference between amino-cytosine and imino-cytosine?

Answer 7

Amino-cytosine (R-(H₂N\C=N)-R) bonds with guanine, imino-cytosine (R-(HN\\C-NH)-R) binds with guanine

Question 8

Base analogues: what are they and what are the examples?

Answer 8

Molecules that can act as substitutes for bases in nucleic acids

The most popular example is 5-bromouracil (5bU) which can act as an analogue for thymine and be incorporated into DNA in place of thymine

Question 9

When is the DNA incorporation of 5bU, the analogue of thymine, not an issue and an issue and what effect does this have on daughter DNA molecules?

Answer 9

If keto-5bU is incorporated, it bonds with adenine as thymine would so there is no issue

The enol-5bU tautomer, which is very common, binds with guanine and this causes a mutation to occur

One out of four granddaughter DNA molecules will be a mutated DNA molecule

Question 10

Deaminating agents: what are they, what are the examples, what are the examples of deaminated bases, and what is the effect on DNA molecules?

Answer 10

Changes the structure of bases from R-(H₂N\C=N)-R to R-(O\C-NH)-R:
Replaces NH₂ with a =O and a =N becomes a -NH

Nitrous acid (HNO₂) and sodium bisulfite (NaHSO₃)

• Adenine may be deaminated into hypoxanthine which bonds with cytosine
• Cytosine may be deaminated into uracil which bonds with adenine
• Guanine may be deaminated into xanthine which inhibits DNA replication
• Thymine has no NH₂ so deamination does not occur

Two out of four granddaughter DNA molecules will be a mutated DNA molecule

Question 11

Difference between adenine and hypoxanthine

Answer 11

Adenine (R-(H₂N\C=N)-R) may be deaminated into hypoxanthine (R-(O\\C-NH)-R) which binds with cytosine

Question 12

Difference between cytosine and uracil

Answer 12

Cytosine (R-(H₂N\C=N)-R) may be deaminated into uracil (R-(O\C-NH)-R) which binds to adenine

Question 13

Difference between guanine and xanthine

Answer 13

Guanine (R-(H₂N\C=N)-R) may be deaminated into xanthine (R-(O\C-NH)-R)

Question 14

Alkylating agents: what are they, what are the examples, and what are the examples of alkylated bases?

Answer 14

Molecules that add alkyl groups to the =O of a base, causing that oxygen to be unable to form hydrogen bonds

Ethyl methane sulphonate (EMS)

O⁶ethylguanine can only form two hydrogen bonds so it bonds with thymine instead of cytosine

Causes transition mutations

Question 15

Intercalated agents: what are they and what are the examples?

Answer 15

A molecule that inserts itself between base pairs

Ethidium bromide

Can make insertion mutations

Question 16

Ultraviolet radiation: what does it do, what are the examples of its effect, and how difficult is the damage to repair?

Answer 16

Ultraviolet rays may hit DNA and cause base dimerisation of DNA molecules

Two adjacent thymine bases may bind to each other instead of forming their base pairs with adenine and this inhibits DNA replication which may be fatal

This is very difficult to repair, which explains why UV radiation is so dangerous

Question 17

Heat: what does it do, what does it cause, and how difficult is the damage to repair?

Answer 17

Heat may cause bases to detach from DNA and give rise to AP (apurinic/apyrimidinic) sites

AP sites will lack a purine or a pyrimidine

This happens very often (~1,000 times a day) and is easy to repair

Question 18

The four types of DNA repair

Answer 18

Direct repair -

Excision repair -

Mismatch repair -

Non-homologous end-joining -

Question 19

Direct repair: what is it, how does it occur, what does it, and how frequent is this method?

Answer 19

Corrects a nucleotide alteration

E.Coli - the ADA enzyme removes alkyl groups from position 4 of T and/or position 6 of G
Humans - MGMT enzyme removes alkyl groups from position 6 of G

Uncommon method

Question 20

Why is UV radiation less dangerous to some species than others?

Answer 20

Some species, for example, E.Coli, have enzymes such as DNA photolyse which directly repair base dimers formed by UV radiation

Other species, humans for example, do not have an enzyme capable of repairing UV damage so suffer from UV radiation

Question 21

Excision repair: what is it and what are the two examples?

Answer 21

The damaged nucleotide is removed and repaired by DNA synthesis

• Base excision repair - the base is removed
• Nucleotide excision repair - A longer piece of DNA containing the altered base is removed

Question 22

How does base excision repair work?

Answer 22

In E.Coli a DNA glycosylase enzyme removes the damaged base, then an AP endonuclease (ie phosphodiesterase) removes the sugar-phosphate backbone that has no base, and then DNA polymerase adds a new nucleotide which then forms phosphodiester bonds due to DNA ligase

Question 23

How does nucleotide excision repair work?

Answer 23

In E.Coli, the UvrABC endonuclease does this

Firstly, the UvrAB trimer attaches to the damaged nucleotide, then the UvrA dimer detaches and the UvrC monomer attaches

Then the UvrBC dimer cuts the segment which is then fully excised by DNA helicase II

UvrB bridges the gap and then the DNA is synthesised by DNA polymerase I and DNA ligase makes the nucleotides form phosphodiester bonds

Question 24

Mismatch error: what is it and how is it recognised?

Answer 24

Correcting errors in DNA replication

The two stands are distinguished by methylation on the strands: (in E.Coli) the parent strands of the new DNA molecules have methyl groups attached to nucleotides and the new daughter strand does not so the correct strand is used as a template

Question 25

What is the process of mismatch repair?

Answer 25

In E.Coli it is recognised by the MutH and MutS enzymes: MutS binds to the mismatched nucleotide and then MutH binds to the closest nucleotide on the daughter strand that is bonded to the nucleotide attached to a methyl group

MutH cuts the DNA and then DNA helicase II removes a certain amount of nucleotides so that the mismatched nucleotide is removed. DNA polymerase I and DNA ligase then synthesise new nucleotides and form phosphodiester bonds

Question 26

Mismatch repair in humans

Answer 26

Not fully understood, methylation is not present, and although MutS binds to incorrect nucleotides, MutH is not present and another protein acts as an endonuclease

Tightly associated with the replication fork, so the daughter strand is recognised during synthesis

Question 27

Nonhomologous end joining: what is it and how does the cell know if it’s a nonhomologous end or the natural end of the chromosomes, and how does the process work?

Answer 27

Corrects breaks in DNA

The presence of telomeres indicates that it is the end of the chromosome, the lack of a telomere shows that the end needs joining

In humans, Ku proteins attach to each side of the nonhomologous end of the DNA and they then attract each other and, once the strands are near each other, DNA ligase forms phosphodiester bonds between them