DNA mutations and DNA repair mechanisms

What is a mutation?

a change in a genetic sequence of an organism

Where do mutations typically appear?

not in every cell - most multicellular organisms are mosaics

What is a point mutation?

the change of a singular nucleotide for another

What are point mutations also often called?

single nucleotide variant (SNV)

State the 2 purines

guanine and adenine

State the 3 pyrimidines

thymine, cytosine, uracil

Transitions

purine → purine

pyrimidine → pyrimidine

Transversions

purine → pyrimidine

pyrimidine → purine

Insertion mutation

extra basepair(s) inserted

Deletion mutation

basepair(s) removed

What is the umbrella term for insertion and deletion mutations?

indel

List 3 diseases caused by point mutations

• sickle-cell disease: GAG is mutated to GTG and glutamic acid becomes valine

• many cancers

• color blindness

List 1 disease caused by insertion mutations

huntington’s Disease - abnormal region of CAG repeats

List 1 disease caused by deletion mutations

duchenne muscular dystrophy

List 1 disease caused by frameshift mutations

crohn’s disease

What is a chromosomal mutation?

where a portion of a chromosome is changed

List the 5 types of chromosomal mutation

• deletion

• duplication

• inversion

• insertion

• translocation

Chromosomal deletion

portion of chromosome is deleted

Chromosomal duplication

portion of chromosome is duplicated and attached

Chromosomal inversion

portion of chromosome is removed, flipped around, and reinserted

Chromosomal insertion

addition of a portion of one chromosome to the end of another

Chromosomal translocation

two portions of two chromosomes are removed and swapped

What is Robertsonian translocation?

a form of translocation that results in one extra long and one extra short chromosome

Where does Robersonian translocation occur?

within acrocentric chromosomes

What are acrocentric chromosomes?

Chromosomes who’s centromeres are located further to one end as opposed to in the middle

List 2 syndromes that can arise as a result of Roebrtsonian translocation

• down’s syndrome

• patau’s syndrome

Polyploidy

a general term for when a cell has more than one set of chromosomes (humans are diploidy)

Polysomy

a case of aneuploidy: having the wrong number of chromosomes

What is triploidy?

where cells have 3 copies of a genome

List 3 trisomy syndromes

• down’s syndrome

• edwards’s syndrome

• patau’s syndrome

What are whole genome duplication caused by?

non-disjunction during meiosis

How has whole genome duplication served as an important mechanism of evolution?

allows duplicated genes to acquire new functions

What organisms are whole genome duplication common in?

plants

Give an example of whole genome duplication in plants

• einkorn wheat has 14 chromosomes

• after hybridisation with a grass tetraploid (four copies per chromosome) -> Durum wheat

• another hybridisation event makes it hexaploid → Bread wheat

How does whole genome duplication cause larger fruits?

more chromosomes need larger nuclei and cells → larger fruit

How many copies of its genome do strawberries have?

8 - octoploid

What is colchine?

a chemical that causes meiosis failure and can induce genome duplication

What can colchine be used for?

commercial farming

Lethal mutations

mutations that can / will result in death

Conditional mutations

a mutation that only expresses itself under certain environmental conditions

Null mutation

results in the absence of coded protein

Dominant negative mutations

genetic mutations that create proteins that interfere with the normal function of wild-type proteins

Suppressor mutations

a second mutation that counteracts the effects of a previous mutation

Silent mutation

mutations, or changes, to a gene's DNA sequence that have no effect on the amino acid sequence coded for by that gene

Why do silent mutations occur?

as a result of the degenerative nature of genetic code

Nonsense mutation

a DNA sequence change that results in a premature stop codon, or nonsense codon, in the transcribed mRNA

What is depurination?

removal of a purine base from a nucleotide

How frequent is depurination in mammalian cells?

18,000x in 24 hours

What is deamination?

removal of an amino acid from a nucleic base

What can a deaminated cytosine turn into?

uracil

Base excision repair (4 steps)

1. DNA glycosylases remove the damaged base

2. AP endonuclease then excises the empty sugar phosphate backbone

3. DNA nuclease adds new nucleotide with correct base

4. DNA ligase seals nick

When is nucleotide excision repair needed

For larger mutations such as dimers caused by UV exposure

Nucleotide excision repair (4 steps)

1. multi-enzyme complex scans DNA and cuts phosphate backbone on either side

2. DNA helicase removes piece of DNA between single strand cuts

3. DNA polymerase fills in

4. DNA ligase seals nick

How does mismatch repair work? (3 points)

1. new strand contains nicks

2. repair complex scans DNA for nicks and removes new strand starting at nick and including wrong base

3. DNA polymerase and ligase fill and seal gap.

List 2 methods of repairing double strand breaks

• non homologous end joining

• homologous recombination

List 2 issues with non-homologous end joining

• introduces mutations

• often involve loss of DNA

List 2 mutations that may arise as a result of non-homologous end joining

• indel

• chromosomes with two/no centromeres

Why is it okay that non-homologous end joining is predominant in humans? (3 points)

• 98% of genome is non-coding

• works within cell cycle

• crucial for VDJ joining (important in lymphocytes)

NHEJ scars count

by age 70 the average somatic cell genome has 2,000 NHEJ scars

What does homologous recombination require?

sister chromatids to use as templates

When does homologous end joining occur?

just after DNA replication

Why is homologous recombination more accurate?

collision of two complimentary strands result in annealing of bases - rapid zippering

What can homologous recombination result in?

a heteroduplex

What is a heteroduplex

a piece of DNA double strand from two different DNA molecules

Homologous recombination: step 1

nuclease resect 5’ strands to create overhangs

Homologous recombination: step 2

overhang invades sister chromatid through base pairing

Homologous recombination: step 3

DNA polymerase extends broken strand using undamaged sister chromatid DNA as template

Homologous recombination: step 4

extended single strand pairs up again with original complimentary strand

Homologous recombination: step 5

gaps are filled

Homologous recombination: step 6

DNA ligase seals nicks

What is CRISPR?

an adaptive immune response to phage attacks in bacteria

How can CRISPR be used for genome editing? (2 points)

• needs synthetic guide RNA to target a specific genome region

• Cas9 protein cuts target DNA

List 2 methods of genome editing using CRISPR

• DNA repairs through NHEJ

  • homologous recombination

State an issue with using NHEJ for genome editing

creates indel mutations

State two advantages to using HDR for genome editing

• allows targeted insertion of DNA

  • allows insertion of tags

List 2 diseases that affect DNA repair machinery

• Werner syndrome

• Severe Combined Immunodeficiency

List 4 symptoms of Werner syndrome

• premature ageing

• osteoporosis

• type 2 diabetes

• hair thinning

State the affected gene in Werner syndrome

WRN RecQ like Helicase

What is the role of the affected gene in Werner syndrome?

double strand break repair (homologous recombination)

State two symptoms of SCI

• increased susceptibility to infection

• B and T cell deficiency

State the affected gene in SCI

ARTEMIS

What is the role of the affected gene in SCI?

double strand break repair in somatic VDJ recombination