Lecture 7 DNA Damage And Mutation

Question 1

Mutation

Answer 1

Any alteration to genetic material (DNA or RNA) that produces heritable change in the nucleotide sequence.

Mutation isn’t: chem damage or mod that causes temp changes in genes or gene function. Hence DNA damage is diff from mutation but can lead to mutation - inherited change in nucleotides. DNA damage needs to be replicated to become an inheritable mutation. Often only half of progeny will be mutant.

DNA damage can be repaired before it results in mutation. DNA damage is proto mutagenic - has potential to lead to mutation.

Question 2

Terminology

Answer 2

Wild-type
Standard form of gene or organism ( in humans wild type is called normal)

Mutant
Altered gene or organism produced by mutation

Forward mutation
A process that converts wild-type to mutant

Reversion
Process converting mutant to wild-type

Phenotype
Description of an organisms appearance

Genotype
Description of the genes of an organism

Question 3

Types of mutation: global change (large scale genomic changes)

Answer 3

Chromosomal aberrations: deletions, insertions, duplications, inversions

Genome rearrangement: redistribution of genetic material between chromosomes (translocations) - often arising from chromosomal breakage

Change in chromosome number: e.g. trisomy of chromosome 21 (downs syndrome) usually arises from mistakes in chromosomal segregation at cell division.

Question 4

Types of mutation: localised changes (affecting a small number of nucleotides)

Answer 4

Base substitutions: point mutations ( single base changes, frame shifts - loss or gain of a nucleotide)

Deletion/insertion (loss or gain of bases usually 2 or more)

Duplication (a sequence is repeated)
Inversion ( sequence inverted)

Translocation/transposition - movement of a piece of DNA from one location to another

Question 5

Base pair substitutions (point mutations)

Answer 5

A mutation that results in the substitution of one base pair for a diff base pair can be :

transition: changes purine or pyrimidine for a pyrimidine e.g. G:C to A:T or T:A to G:C
or
transversion: changes purine for pyrimidine or pyrimidine for a purine e.g. G:C to (C:G or T:A) or T:A to (G:C or A:T)

Question 6

Possible outcomes of base pair substitution

Answer 6

Mutation:

Silent or samesense - no effect on amino acid sequence

Missense- result in amino acid substitution

Nonsense - change amino acid to stop codon

Read-through - changes stop codon to an amino acid

Frame shift - base pair deletion or insertion in gene induces shift in reading frame changing the protein sequence downstream of the change often resulting in truncated protein

Question 7

Effects of point mutation on proteins

Answer 7

No effect : samesense/silent (DNA polymorphism: protein is unaffected by DNA variation introduced)

Missense: protein polymorphism an amino acid change may not affect protein function

Change of function: missense, small deletions > altered protein

Loss of function: missense, nonsense and frame shifts > protein no longer made

Question 8

Terminology

Answer 8

Mutagen: chemical or physical agent that causes mutation

DNA damage: chemical lesions in DNA

DNA repair: removal of DNA lesions

Mutation: molecular process by which heritable changes arise

Spontaneous mutation: genetic changes that arise naturally during the life of an organism.

Induced mutation: genetic changes caused by specific mutagen

Question 9

Categories of damage

Answer 9

Adduct/lesion used interchangeably to refer to DNA damage

Affecting ss of DNA:
Adduct or lesion (1 or 2 nucleotides altered e.g. methylated bases) a nick or mismatch ( e.g. G:T)

Affecting both strands of DNA:
Replicated adduct (a gap opposite the adduct) a chromosome break or interstrand crosslink

Question 10

Classification of mutations : spontaneous

Answer 10

Spontaneous:

Endogenous factors:
Loss of bases
or amine groups from bases
Mutations from damage by metabolic products(e.g. reactive oxygen species ROS - can also be considered induced damage)
Fixation of mismatches and other mistakes by DNA polymerases

Question 11

Classification of mutations: induced

Answer 11

Damage from external (exogenous) factors:

Radiation e.g. UV, X rays, gamma rays alpha and beta particles from nuclear decay

Alkylation of bases

Crosslinking agents
Intercalated molecules

Question 12

Spontaneous DNA damage: depurination

Answer 12

Glycosidic bond between base and sugar is cleaved by hydrolysis resulting in apurinic or apyrimidinic (AP) site aka abasic site. Loss of A or G (purine) is most common. A human cell typically loses several thousand purines a day

Question 13

Spontaneous DNA damage: Deamination

Answer 13

Amine groups on the rings of the bases are susceptible to spontaneous oxidation to aldehyde groups, a process known as Deamination. This alters the pairing properties of the bases e.g. cytosine deaminates to uracil which can base pair with adenine ( gives rise to G:C to A:T transition mutations if unrepaired)

Question 14

Spontaneous DNA damage: Tautomeric shifts

Answer 14

Bases in DNA can occur in several forms, tautomers, which differ in their position of atoms and in bonds between the atoms. The keto (G/T) or amino (A/C) form of a base is normally present in DNA) whereas the corresponding enol and imino forms of the bases are rare. DNA bases can isomerise and diff isomers have diff base pairing properties. These changes are a significant source of spontaneous mutation.

E.g. guanine undergoes tautomeric shift to it’s rare enol form (G*) prior to replication. In its enol form it pairs with thymine but post replication it reverts to normal keto form. Another round of replication results in a G:C to A:T transition mutation in one of its progeny.

Question 15

Induced DNA Damage: endogenous oxidative damage

Answer 15

Biggest danger to DNA in cells are the products of the oxidation process particularly oxygen radicals - uncharged molecules with a single unpaired electron

E.g. thymine glycol formed by hydroxyl radical attack of thymine blocks DNA replication

E.g. formamidopyrimidine (FaPy) formed by hydroxyl radical attack of guanine breaking the imidazole ring

Question 16

Damage limitation

Answer 16

Avoiding damage is better than repairing it. Oxidation of FADH2 on flavoproteins leads to production of damaging oxidants: hydrogen peroxide and superoxide.

Catalases (Kats), peroxidases ( e.g. alkyl hydrogen peroxide reductase, Ahp) and superoxide dismutases (SODs) minimise the accumulation of these two oxidants.

Glutathione is a tripeptide used as a cellular antioxidant to mop up ROS (reactive oxygen species)

Question 17

Induced DNA damage: Radiation

Answer 17

Indirect effects: particle interactions with other molecules which then interact with the DNA e.g. from radiolysis of water to give hydroxyl radicals. Hydroxyl radicals generate damaged bases. The production of clustered legions can also lead to chain breaks.

Direct effects: particle imparts its energy directly to the DNA molecule breaking the bonds that hold the sugar phosphate backbone together. UV has a direct effect on DNA but most is absorbed by the ozone layer.

Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main products of UV radiation. They are formed at adjacent pyrimidines, resulting in covalently fused dimers. The lesion interferes with normal base pairing and can yield base substitutions. Double helix is distorted hence this type of damage is referred to as a bulky lesion. In a CPD a 4 membered cyclobutane ring is formed between two adjacent pyrimidines. The following lesions are formed preferentially in the following order T<>T > T<>C > TC(6-4) > c
C<>T > C<>C > TT(6-4)

6-4 photoproducts
The C6 carbon of one pyrimidine covalently links with the C4 of the adjacent pyrimidine. As with a CPD the DNA backbone is distorted resulting in a bulky lesion. 5’- methylcytosine found in higher organisms never forms 6-4 lesions. The 6-4 photoproducts TC(6-4) and CC(6-4) and less frequently TT(6-4) are observed in UV irradiated DNA. CT(6-4) photoproducts do not occur.

Question 18

Induced DNA damage: chemical mutagens

Answer 18

Both natural and artificial agents causing a range of lesions in DNA including some of the most potent carcinogens known

Alkylating agents: add methyl or ethyl groups to bases. The addition to the aromatic rings can alter the pairing properties and so are often called miscoding lesions. Some even block replication (i.e. non coding regions) Benzo(a)pyrenethe carcinogenic ingredient in chimney soot, cigarette smoke and car exhausts is metabolised to an epoxide that acts on the N2 group of guanine

Question 19

Mutagens produced by metabolic activation

Answer 19

Many mutagens require bioactivation to electrophiles that bind covalently to DNA. Acetylaminofluorine (AAF) an aromatic amine group originally used as an insecticide is converted to an alkylating agent by esterification. Aflatoxins produced by Aspergillus flavus and commonly found as a contaminant of mouldy peanuts is metabolised to an oxide derivative that acts on the N7 of guanine

Question 20

DNA crosslinking

Answer 20

Pyrimidine dimers are an example of intrastrand crosslinking. Certain chemicals are capable of covalently joining two bases in complementary strands to form and interstrand crosslink. DNA crosslinks prevent separation of the two strands blocking transcription and DNA replication. Interstrand crosslinks are typically produced by bifunctional agents e.g. nitrogen mustard - has 2 nucleophilic methyl groups: mitomycin C and psoralens natural products derived from a fungal source

Question 21

DNA intercalating agents

Answer 21

DNA double helix is partly held together by stacking interactions. Some chemicals are capable of intercalating (slipping between) the stacked DNA bases e.g.ethifium bromide, acridine orange, proflavin. Intercalation can cause single nucleotide insertions and deletions and block replication and transcription.