The nature and mechanism of mutations in the genome.

Question 1

Define Genetic Variation

Answer 1

Differences in the DNA sequence of individuals in a population: variations can arise de novo (somatic) or be inherited (germline).

Question 2

Describe mutations

Answer 2

Mutations are random-event and arise through environmental agents (UV, chemical, viruses) or mutations of repair genes. Mutations are not driven; natural selection selects mutations already present in a population. However they can be repaired.

Question 3

What are the external causes of mutation?

Answer 3

Radiation (UV & thymidine dimers), chemicals that cause depurination, demethylation or deamination.
Mutagens can be base analogs (5-bromouracil)
Cause structural changes (intercalating agents) or act indirectly (cell to produce other chemicals that have n effect)

Question 4

What are the internal factors that cause mutations?

Answer 4

Internal factors are generally replication errors.
~6 billion bases (ACGT) are copied every cell division: this replication must be faithful after every cell cycle otherwise deleterious mutations accumulate which is incompatible with life.
Replication however cannot be perfect as there would be no evolution without it. Mostly corrected by proof-reading errors.
Most mutations are loss of function.

Question 5

What is a tautomer?

Answer 5

Tautomers are two molecules with the same molecular formula but different connectivity, they are constitutional isomers, which can interconvert in a rapid equilibrium.

Question 6

Describe the tautomeric properties of DNA bases.

Answer 6

Stable DNA bases exist in keto form (T and G) and amino form (A and C) transitions then occur to unstable forms.

Question 7

Which bases are enol in their unstable form?

Answer 7

T and G

Question 8

Which bases are imino in their unstable form?

Answer 8

A and C.

Question 9

Can unstable base forms create bonds?

Answer 9

Yes, but they are also unstable. T:G and A:C. The instability ensures that complementary binding rules is always upheld.

Question 10

What are mutagens?

Answer 10

They are external factors that increase the rate of mutation above the spontaneous background rate.

Question 11

What are the mechanisms of mutagenesis?

Answer 11

Deamination (spontaneous and induced) (Nitrous aced = induced.
Alkylation = mustard gas, EMS and EES
Depurination
Hydroxylation/ Oxidation = hydroxylamine
Base analogs = 5-Bromouracil/ 2-aminopurine
Intercalating agents (ethidium bromide, green fluorescein)
UV radiation
Ionizing radiation.
Proflavin = acridine

Question 12

What does deamination do to cytosine?

Answer 12

Produces Uracil

Question 13

What does deamination do to 5-methylcytosine?

Answer 13

Produces Thymine.

Question 14

How can deamination occur?

Answer 14

Spontaneously or Induced.
Methyl-cytosine deamination into thymidine is a spontatneous reaction with C:G and T:A binding. HNO2 is a potent deaminator.
Hypoxanthine is similar to imine (A) A:T pair becomes G:C
Xanthine is similar to G (enol) G:C pair becomes A:T
- some of these reactions can change sequences, inducing functional consequences.

Question 15

How much of the genome is CpG islands?

Answer 15

0.8%

Question 16

What size are CpG islands?

Question 17

Where can CpG islands be found?

Answer 17

They are associated with the 5’ end of genes (in 40-50% of known genes). Up to 10% of CpG are methylated on the cytosine molecule.
Methylation can result in repression of expression (tissue-specific methylation in restricted genes for example)
Deamination of the C results in a T nucleotide (so CpG becomes TpG). The effect of these changes is variable, from nothing to chronic haemolytic anaemia, leukaemia and some forms of cancer.

Question 18

Give examples of alkylating agents.

Answer 18

Mustart gas, EMS and EES.

Question 19

What do alkylating agents do?

Answer 19

Alkylation is a natural genetic process such as methylation or acetylation, however alkylating agents induce alkylation where it should not occur:
They donate alkyl groups to other molecules
Cause transitions, transversions, frameshifts and chromosome aberrations.
Alkylation of bases can change base-pairing properties (GC AT) and alkylation can also activate errors during repair processes.

Question 20

What is depurination and hydroxylation and how does it affect DNA?

Answer 20

Hydrolysis reactions remove purine (A/G) rings by cleaving the N-glycosidic bond that holds them to the sugar. An adenine-containing nucleotide is usually incorporated across from the depurinated one during the next round of replication.
Hydroxylamine hydroxylates the amino group of cytosine and can lead to G:C -> A:T transitions.

Question 21

What are base analogs?

Answer 21

Similar structures to regular DNA bases and when they are incorporated into DNA they increase the frequency of mis-pairing. Not all are mutagenic: AZT is a thymidine analog.
Examples include: 2-aminopurine: adenine analogue which pairs with cytosine.
5-bromouracil: thymine analogue pairs with guanine in enol form, binds to adenine in keto form.

Question 22

How do intercalating agents affect DNA?

Answer 22

They do not directly affect the bases, but their position..
They are thin, plate-like hydrophobic molecules that insert themselves between adjacent base-pairs. They are generally +vely charged molecules. Mutagenic intercalating agents cause insertions during DNA replication (changing the distance between bases)
Loss of intercalating agent can result in deletion. Examples include proflavin and ethidium bromide.

Question 23

How does UV radiation cause mutagenesis?

Answer 23

Hydrolysis of cytosine to a hydrate which may induce mis-pairing during replication copied in subsequent rounds of replication.
Causes cross-linking of adjacent thymine forms thymidine dimers (6% of genome): blocking DNA replication and activating DNA repair mechanisms.
Ionizing radiation can break covalent bonds between bases and cause chromosome mutations.
UV (254-260nm) causes purines and pyrimidines to form abnormal dimer bonds and buldges in the DNA strands.
Ionizing radiation has a cumulative effect and kills cells at high doses. Low level continual exposure: mutation accumulation, increasing likelihood of deleterious outcome on the cell.