DNA Mutation and Human Health 1

Lecture Overview on DNA Mutation and Human Health

• Instructor: Dr. Andrew Nesbit

• Content Outline:

  • What causes mutations?

  • DNA damage and repair mechanisms.

  • Types of mutation and their consequences on human health.

Definition of Mutation

• Mutation: A structural change in genomic DNA that can be passed from a cell to its daughter cells.

Importance of Mutation in DNA

• Changes in DNA sequence lead to changes in the coded proteins, which is central to molecular biology's central dogma.

• The Central Dogma of Molecular Biology is a theory that states that the flow of genetic information in biological system travels one way, from DNA to RNA to Protein, or directly from RNA to protein.

Causes of Mutations

• Main Causes:

  • Errors during DNA replication or recombination.

  • The major source is failure to repair DNA damage, which occurs due to:

    • External agents causing DNA damage.

      (Radiation, Mutagens, etc.)

    • Internal cell chemistry producing toxic compounds that damage DNA.

      (e.g. Reactive Oxide species)

External DNA Damaging Agents

Ionizing Radiation

• Sources: Gamma rays and X-rays.

• Effects: Can cause breaks in the DNA sugar-phosphate backbone.

• Can be done directly or indirectly via production of free radicals

Ultraviolet Radiation

• Source: Mainly UV-B rays from sunlight which penetrate ozone layer

• Effects: Causes thymine dimers through cross-linking of adjacent thymines (hydrogen bonds to adenine broken)

Environmental Chemicals

• Common agents include:

  • Hydrocarbons from cigarette smoke.

  • Aflatoxins from plants/microbes (e.g., Aspergillus species).

  • Various chemotherapy agents like Ethidium Bromide.

Cancer-Causing Chemicals in Tobacco Smoke

• Examples include:

  • Tar, arsenic, benzene, cadmium, formaldehyde, polonium-210, chromium, 1,3-butadiene, polycyclic aromatic hydrocarbons, nitrosamines, acrolein, acetaldehyde.

Internal DNA Damaging Agents

Depurination and Deamination

• Depurination: Loss of adenine or guanine; occurs ~5000 times daily in a typical cell.

• Deamination: Conversion of cytosine to uracil; occurs ~100 times daily.

Reactive Oxygen Species (ROS)

• Highly reactive sub-type of free radicals which contain oxygen

• By-products of mitochondrial metabolism, e.g., superoxide anions, hydrogen peroxide (H202), OH hydroxyl radicals.

• Have important cellular functions such as roles in cell signalling and homeostasis. Cell has natural antioxidant molecules/defences which keep levels in check (normally)

• Effects:

  • Attack DNA sugar-phosphate backbone causing single-strand breaks.

  • Damage bases, transforming them (8-oxo guanine, thymine glycol), increases the risk of mutation

• This can result in Non-Enzymatic Methylation

  • This is the addition of Methylation to Nucleotide bases, changing the way they base pair (e.g. Gaunine base pairing to Thymine)

Repair Mechanisms for DNA Damage

• Four main types:

  • Base excision repair (BER).

  • Nucleotide excision repair (NER).

  • Homologous recombination (HR).

  • Non-homologous end joining (NHEJ).

• Each method is used depending on the type of damage

• These mechanisms are a part of the DDR (DNA Damage response). It is a collection of different proteins, mechanisms, processes & biochemical pathways that function to detect DNA damage (lesion-specific sensor proteins) & mutations, and repair them, or cause apoptosis if the damage is beyond repair

Base Excision Repair (BER)

• Targets: Oxidized, alkylated, or deaminated bases.

• Key enzymes involved:

  • Uracil-N-glycosylase, DNA polymerase, DNA ligase.

• Uracil-N-Glycosalase will break the bond between the changed base and the sugar-phosphate backbone, apyrimidinic or apurinic endonucleases will cleave the phosphodiester bonds in the DNA backbone where that base was, creating a free hydroxyl and phosphate group. DNA polymerase will then read the opposite strand and synthesise the correct nucleotide base which will be added to the correct position in the sequence, and DNA ligase will repair the backbone by catalysing the formation of bonds between the 5’ and 3’ ends

Nucleotide Excision Repair (NER)

• Used to repair bulky DNA lesion (needed for thymine dimers due to Uv-B)

• The process includes scanning DNA for lesions, denaturing DNA, removing damaged fragments & synthesising new ones to replace them.

  Key proteins: UvrA, UvrB, UvrC, and UvrD.

• UvrAB complex scans DNA and arrives at lesion where UvrB binds to it, UvrA leaves and UvrB denatures and bends the DNA at the lesion. UvrC arrives, binds to UvrB and hydrolyses the phosphodiester bonds at the 5’ and 3’ ends of the lesion. UvrD, a helicase, arrives and displaces UvrC. UvrD takes the lesion (12-13mer in Prokaryotes, 24 to 32 in eukaryotes), DNA poly I arrives, binds to the DNA sequence and repairs the gap. DNA ligase then repairs the backbone.

Homologous Recombination & Non-Homologous End Joining

• Involved in larger DNA lesions/damage (Double Stranded DNA breaks, (DSB)

• DSB are the most cytotoxic type of DNA damage, leading to apoptosis, loss of chromosome segments and stopping replication

  • (cells that have large DNA damage like this will suffer arrest of the cell cycle after one of the G1 or G2 checkpoints)

• Non-Homologous End Joining

  • Is used before Synthesis phase of cell cycle, as there is no copy to use to repair the break (as both strands are broken)

  • Occurs in all cells in the body

  • Doesn’t involve DNA synthesis

  • Quick method for DNA repair, method works to “glue” or stick the broken DNA double-strand back together

  • Can result in loss or gain of nucleotides within gap being repaired, can change expressed proteins if present in coding regions (frameshift mutation)

  • During breaks, nucleotide overhangs can occur (resection, single strand overhangs). Protein dimers (KU70/80) is recruited at these broken ends, which recruits DNA-Pks, which recruits & phosphorylates artemis, resulting in the trimming of these overhangs. Ligase IV is then recruited along with XRCC4, etc. which results in fixing of the break

Homologous Recombination

• Is used after synthesis phase of cell cycle, as there is a copy of the DNA strands to use as a template

• Relies on DNA synthesis & cell division

• Through Cell Division/replication, the undamaged chromosome is read & new fragment is synthesised

• Results in a complete and seamless repair of damaged section of DNA

• Slower process, but much lower risk of mutation occurring

• This process involves reading the fully intact copy of the DNA double-strand produced during DNA synthesis, producing an identical strand of DNA pre-break. This method can result in the crossover of the DNA double strands, or simply annealment of the newly synthesised strand to the broken one.

Consequences of DNA damage

• Cytotoxic to the cell

  • The cell will recognise the damage via DDR, and attempt to repair it. If repair is not possible, apoptotic pathways will be initiated and the cell will undergo apoptosis

• Result in Replication of Damaged DNA

  • If the DNA is damaged in a coding region, a mutated protein will be expressed, resulting in a diseased state

Types of Mutations

Small Scale Mutations (Point Mutations)

• Missense Mutations: Substitution of one amino acid for another when a nucleotide base is changed, resulting in a different codon

  • Conservative Mutation = No change in Protein Function (Substituted amino acid has similar/same properties as replaced one)

  • Non-Conservative Mutation: Change in Protein Function (Substituted amino acid possesses different properties than replaced one)

  • Most common type of mutation: 47%

• Nonsense Mutations: Creation of a STOP codon leading to truncated/non-functional proteins.

  • UUA, UAG, UGA

  • Less common than missense

  • Results in shortened/truncated protein, likely major consequences to protein function and structure, unless stop codon is formed extremely close to the intended one (unlikely)

• Splice Site Mutations: Alteration in Base pairs at Exon/Intron Boundary

  • Removal of an exon: Results in truncated protein

  • Inclusion of an intron: Alters protein coding sequence

  • Disruption of splice site silencers & enhancers: prevents splicesome binding

  • All of these can affect protein structure, function and produce non-functioning proteins

• Frame-shift Mutations: Caused by insertion or deletion of nucleotides

  • Results in change of reading frame in a protein, causing every codon after it in the sequence to change

  • this can potentially result in huge amounts of incorrect amino acids (missense) being added to the protein chain, causing catastrophic effects on protein function & severe phenotypic effects/changes

• Silent Mutations: Do not cause a change in the amino acid sequence due to redundancy in the genetic code.

  • Genetic code is degenerative, meaning multiple codons can code for the same amino acid

  • If a mutation occurs in a codon, changing it to another codon that codes for the same amino acid, then no change in protein function or structure occurs

Large Scale Mutations

• Involves deletions or duplications of chromosomes or chromosomal segments.

Frequency of Known Mutations in Humans

• Distribution of mutation types (in %):

  • Missense: 47%

  • Nonsense: 11%

  • Splice-site: 10%

  • Regulatory: 1%

  • Gross deletions: 5%

  • Small deletions: 16%

  • Gross insertions: 1%

  • Small insertions: 6%

  • Others: 3%

Consequences of Mutations on Human Health

• Functional Effects Considered:

  • Loss-of-function (most common).

  • Gain-of-function alterations.

  • Dominant-negative effect.

Loss of Function

• Definition: Protein product has reduced or no function.

• Haploinsufficiency: A specific loss-of-function where 50% of normal protein is inadequate for normal function.

• Examples of diseases:

  • Hereditary hemorrhagic telangiectasia (HHT).

  • Alagille syndrome Type 1.

  • Tomaculous Neuropathy

  • Multiple Exostoses type 1

Gain of Function

• Typically Changes the way a protein responds to regulatory signals:

  • Expressed at wrong time in wrong location

  • Interacts with other cellular components, producing pathogenic effect

  • Inability to respond to negative signals

• Mutations are unlikely to give a protein a radically different function. however, a chromosomal rearrangement can result in a chimeric gene (new function)

• Examples:

  • Receptor permanently on: Ras oncogene or BCR-ACL: Chronic Myeloid Leukaemia

  • Chimeric gene: GNAS1: McCune-Albright Disease

  • Overexpression: NROB1: Male to Female Sex Reversal

  • Acquires New Substrates: PI: Lethal Bleeding Disorder

  • Protein Aggregation: HD: Huntingtons Disease

Dominant-Negative Effect

• Product of a defective gene interferes with normal protein function.

• Example: Marfan syndrome affecting FBN1 gene, codes for fibrillin 1. results in long limbs, heart & aortic defects

Key Takeaways

• Mutations in DNA lead to changes in protein coding, affecting human health.

• Mutations arise from both internal and external factors, with cellular mechanisms in place to repair them.

• Unrepaired mutations can lead to cell death through apoptosis, or result in mutated proteins leading to various health consequences.

• Changes in DNA sequence results in a
  change in the coded protein.
  ② This can have effects on human health
  ③ Mutations to DNA can be caused by internal
  and external factors.
  ④ The cell has various mechanisms to try to
  repair the mutation.
  ⑤ However the mutation may be lethal and
  the cell will die by apoptosis.

  ⑥ Otherwise the cell will survive but will have a
  mutated protein – this leads to
  consequences to health.
  ⑦ Mutated protein can have
  ① Loss of function
  ② Gain of function
  ③ Dominant negative
  ⑧ Each of these can lead to diseases