Lecture 2 - basis of mutations

genetic variation

differences in our genes

phenotypic variation

differences in our observable characteristics

dominant alleles

always hsow their effect, will be expressed even if individual is heterozygous (only has 1 copy of the allele)

recessive alleles

only show their effect if individual is homozygous (has 2 copies of the allele)

acquired mutations

not inherited, acquired through DNA damage

causes of DNA damage

environmental agents

bi-products of normal metabolism

spontaneous damage

snp

single nucleotide polymorphisms - small mutations in 1 codon of DNA. may significantly impact protein structure, function and activity. e.g. substitutions, insertion or deletion

environmental agents

e.g. UV, radiation, natural/synthetic genotoxic chemicals

exposure to radiation

releases electrons from atoms/molecules, causes ionisation. e.g. x-rays, gamma rays, radon gas etc.

cause DNA strand breakage (single or double).

single strand breakage

easy to repair

double strand breakage

harder to repair. mis-repairing leads to mutations/chromosome aberrations. normally leads to apoptosis

2 ways radiation can damage DNA

indirectly (water absorbs large amounts of radiation, becomes ionised and becomes free radicals e.g. hydroxyl radical which damage the backbone of DNA)

directly (radiation collides directly with DNA itself causing ionisation)

lower energy examples of radiation

visible light, UV, radio waves, infra-red radiation, electroagnetic fields

UV mutagenic component

UVB (280-320nm). absorbed by pyrimidine bases, causes dimerisation. can impede DNA polymerase and arrest replication by causing bulking/distortion of DNA when 2 pyr bind.

2 types of pyrimidine mutations caused by UVB

cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4)
pyrimidone photoproducts.

CPDs are 85% of mutations in UV-irradiated DNA and primary cause of melanoma

what do the pyrimidine dimers target

proto-oncogenes and tumour suppressor genes e.g. p53

p53 normal role/mutation in UV mutations

respond to damage from UV radiation. proteins from p53 gene accumulate in nucleus of cells exposed to UV, delays cell cycle to allow DNA repair or apoptosis initiation. mutations means loss of control functions.

genotoxic chemicals (natural/synthetic)

aldehydes, polycyclic aromatic hydrocarbons, nitrosamines, asbestos minerals. lead to mutations e.g. single strand
breaks, substitutions, insertions and deletions

benzopyrene

natural. found in fossil fuels and formed by incomplete combustion of carbon. metabolism (by CYP1A1) forms BPDE which covalently binds to DNA (N2 position of guanine) and forms an adduct. sits in middle of G and C bases.

impedes movement by DNA polymerases which may try to remove the guanine —> base misincorporation

BPDE targets

targets the p53 gene, prevents DNA pol from functioning.

mutations in CYP1A1 consequences in benzopyrene

increasing enzyme activity means more adduct formation and potentially more carcinogenic.

p53 general role

guardian of the genome. alerts cells to dna damage. dna is either repaired or the cell dies. without p53, cells can divide unchecked and may lead to cancer.

high levels of p53 consequence

excessive apoptosis can accelerate aging process

reactive oxygen species

superoxide, peroxide, hydroxyl radical etc. unpaired electron.

can react with backbone of DNA and cause strand breaks

how ros are formed in etc

etc produces atp via oxidative phosphorylation. electrons leak and react with water forming ros. if they escape detoxification processes can cause cellular damage.

as mitochondria gradually deteriorate, ros production increases, over time the mitochondria break down leading to reduction in ATP production (energy deprivation).

mitochonridal dysfunction is characteristic of aging/chronic diseases.

why is mitochondrial dna susceptible to attack by ros

close to innner mito membrane where etc and ros are found.

lack protective proteins such as histones so are more exposed to ros attack

exogenous sources of ros

ionising radiation, pollutants, tobacco, smoke, drugs, xenobiotics

examples of mutations caused by ros

single strand breaks, base crosslinking, base modifications, loss of bases (abasic sites)

spontaneous dna damage

damage occurs independently of environmental factors. e.g. errors in replication. if no dna repair mechanisms fix it: base pair mismatches, single strand breaks, insertions, deletions, depurination and deamination

difference between mutation and polymorphism

any change in DNA sequence in an allele that changes it to a rare/abnormal variant (mutation <1% of population, polymorphism >1% of population).

2 types of base substitution

transition (purine to purine/pyr to pyr). transversions (purine to pyr or pyr to purine).

example of a G to T conversion

transversion. can occur in p53 gene.

if guanine base damaged from BPDE adduct, dna pol will often insert an adenine opposite the damaged guanine, and later a thymine is replaced opposite the new adenine: transversion. will lead to damaged p53.

further subdivisions of base substitution mutations

missense, nonsense, silent

missense mutation & example

mutation results in codon for a different amino acid. can result in non-functional protein.

e.g. sickle cell anaemia (beta chain of Hb changed from GAG (glutamic acid) to GTG (valine) which is less polar so Hb becomes less soluble in low oxygen, distorts RBCs to sickle shape.

nonsense mutations

mutations that result in premature stop codon. short, truncated protein forms. e.g. 15-30% of all inherited diseases: cystic fibrosis, haemophilia, thalassaemia.

silent mutations

pont mutations that dont alter the phenotype of the individual, can occur in non coding regions or within exons. (if multiple different codons code for the same amino acid)

types of point mutation

substitutions, insertions, deletions

frameshift mutations

shift the grouping of the bases all the way along the chain, changing the code of the amino acids resulting in a short non functional protein

tay sach’s disease

autosomal recessive fatal disease of nervous system. insertion of 4bp in exon 11 of hexoseaminidase gene on chr15 —> frame shift mutation, hexosaminidase A deficiency (enzyme crucial to CNS).

common amongst Ashkenazi Jews (1 in 27 carrier).

Gilbert’s syndrome

relatively harmless mutation. very common.

faulty UDP-glucuronosyl transferase 1A1 gene on chr2.

normally, causes conjugation of bilirubin with lipophilic molecules, makes bilibrubin water-soluble (easy to excrete)

if mutated, liver cant remove bilirubin, builds up in blood —> yellow skin and eyes.

caused by dinucleotide (TA) insertion in TATA box of enzyme gene

deletions &example

frameshift mutations also occur.

beta-globin gene. cysteine in codon 39 is deleted - reading frame is completely altered. results in blood diseaes such as beta thalassaemia

large scale deletions

Jacobsen Syndrome, deletions of genes in chr11

large scale insertion

portion of one chromosome is deleted from one site and inserted into another

common in haemophilia A

dysfunctional factor VIII clotting enzyme.

inversions

revering orientation of chromosome section

also in factor 8 gene in haemophilia A resulting in dysfunctional protein

translocations

different chromosomes swap genetic material

can result in gene fusion. common in cancers.

90% of CML patients: extension of chr9, shortening chr22: BcrAbl, Philadelphia translocation.

leads to uncontrolled cell growth as genes are involved in growth suppression and are overactivated after fusion

gene amplification

many copies of a gene are expressed.

e.g. overexpression of erbB-2 gene chr17 —> overexpression of Her2 receptor

important role in development and growth of 20% of breast cancers, important biomarker, easy to target

beneficial mutations

lactose tolerance, sickle cell

lactose tolerance

lactase gene used to be suppressed after infancy.

lactase mRNA production regulated by OCT-1 enhancer.

SNPs: C-T mutation in OCT-1 enhancer, increases its affinity for the enhancer region meaning lactase gene is always expressed so milk can be digested throughout life.

useful mutation as drinking milk prevents osteoporosis, nutritious etc.

sickle cell disease beneficial mutation

required 2 mutated copies of haemoglobin for disease.

if individual is heterozygous, can protect against malaria.

female mosquito cannot replicate in blood if some RBCs are sickle shaped. so some resistance against the disease is generated.

lethal muations example. tay sachs disease

ACAT insertion —> hexosaminidase A deficiency.

HexA degrades GM2 Ganglioside (lipid in the nervous system), so deficiency in HexA leads to accumulation of lipid in neurons.

normal development for 6 months but will progressively lose ability to see, hear, move and function (dementia, paralysis etc) - by age 2 seizures will develop, fully disabled, death by age 5.