12. DNA damage and repair

double-stranded DNA structure

• Name the 3 main components

• Name the DNA bases

• How many hydrogen bonds between the DNA bases when bonded?

• Phosphate group, Pentose sugar (deoxyribose) & Nitrogenous base

• Adenine, Thymine, Cytosine, Guanine

• A & T have 2 hydrogen bonds, C & G have 3 hydrogen bonds

Genome stability

• How many cells are you roughly made up of?

• How many DNA damage events can an individual cell experience per day?

• When does DNA damage start?

• 32 trillion cells

• An individual cell can experience up to 1 million DNA damage events per day.

• DNA damage begins from the moment you were a single cell and is constantly happening.

Common types of DNA damage

• Name 4 examples of sequence changes.

• Structural changes include localised structural changes and large structural changes, name the examples for each of these (3).

• Name the consequences of these changes (1st consequence has 3 types, 2nd consequence is 3 related forms of alteration to bases)?

Sequence changes

• Reactive oxygen species

• Thymine dimers

• Deamination

• Depurination

Structural changes

• Localised structural changes

  • Single strand breaks

  • Double strand breaks

• Large structural changes

  • Chromosomal rearrangements e.g. translocations

Consequences

• Point mutations

  • Silent (different DNA sequence but same amino acid)

  • Missense (different amino acid results)

  • Nonsense (forms premature stop codon)

• Insertions/deletions/duplications

Reactive Oxygen Damage

• What is reactive oxygen damage?

• What can ROS be generated by?

• What do ROS react with and what does this do to it?

• What can ROS also attack and cause?

Under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids and DNA, leading to fatal lesions in the cell that contribute to carcinogenesis.

• Can be byproducts of cellular metabolism or generated by radiation exposure.

• Reactive oxygen species (ROS) react with DNA bases, changing their chemistry (oxidised bases) and disrupting base pairing.

• Can also attack the DNA backbone, causing breaks.

Thymine Dimers

• What are thymine dimers?

• What type of bond is formed?

• What do thymine dimers inhibit?

• A covalently bonded complex of two adjacent thymines on a single strand of DNA.

• Covalent.

• Inhibits DNA replication.

Deamination

• What is deamination?

• The removal of an amino group from a molecule.

• The loss of the amine (NH2) group from cytosine bases

• Changes to Uracil (U) – not found in DNA

• 100 bases per cell per day

• Affects DNA replication

• Polymerase can mistake uracil for thymidine (T)

• Causes and G-A switch in the new sequence (incorrect)

• Can be repaired via base excision repair (BER)

Depurination

• What is depurination?

• How many bases are lost per cell per day by depurination?

• What are the bases removed from?

• What does depurination consequently cause a loss of and what does it decrease?

• What can depurination be repaired by?

• Process in which the purine base of a DNA molecule is lost - spontaneous loss of adenine or guanine bases.

• 5000 bases lost per cell per day.

• Become removed from the DNA backbone.

• Causes a loss of genetic information, decreases stability in this region – generally not good.

• Can be repaired via base excision repair (BER)

Single strand breaks (SSBs)

• What type of structural change are SSBs?

• What can they be caused by?

• What are they common for?

• What do they interfere with?

• Are they difficult to repair and how are they repaired?

• What can they develop into if not repaired?

• Localised structural change.

• Naturally occur during many forms of DNA repair (e.g. BER) and can also be caused by other factors.

• One of the most common lesions in cells .

• Interferes with DNA replication and transcription.

• Easily repaired by the cell, using a range of different SSB repair pathways.

• Can develop into more severe damage if not repaired efficiently - E.g. become a double strand break.

Double strand breaks (DSBs)

• What type of structural change are SSBs?

• How are they caused (3)?

• How serious are they?

• What often happens as a result of DSBs (2)?

• What response is triggered?

• How are DSBs primarily repaired (2)?

• What can happen if DSBs are not resolved?

• Localised structural change.

• Caused by:

  • Ionizing radiation and carcinogens can directly break the DNA backbone on both strands.

  • Can be caused by unresolved stalled replication forks.

  • Also occur naturally during meiosis and recombination.

• Very serious.

• Results of DSBs:

  • Cell cycle is often arrested, disrupting replication/transcription.

  • Can lead to large genome rearrangements .

• A significant DNA damage response is triggered .

• Repaired primarily through non-homologous end joining (NHEJ) or homologous recombination (HR).

• If not resolved, normally triggers apoptosis - If significant, can impact tissue function.

Large structural changes

• Failure to repair DSBs properly can cause large chromosomal abnormalities

• Or improper telomere regulation

• Deletion of entire chromosome regions or translocations

• This is where DSBs at different genomic sites are incorrectly joined together

• Causes genomic regions to be in the incorrect place

• E.g. t(9;22) translocation, also known as the Philadelphia chromosome

• Causes many problems..

Sequence changes

Damage or incorrect repair can lead to point mutations

• Silent (different DNA sequence but same amino acid)

• Missense (different amino acid results)

• Nonsense (forms premature stop codon - PTC)

Sequence changes

DNA also experiences insertions/deletions/duplications

• Nucleotides are gained or lost from a sequence

• Causes a frameshift

• Can cause STOP codons to be abnormally present

• Can disrupt the whole sequence (frameshift)

• Or add additional information

Mutations in disease

• Genome instability and unresolved damage lead to mutations

• Mutations in coding regions of the genome lead to altered protein function •

• Can cause a loss of function

• Can cause abnormal gain and functions (dominant negatives) - proliferation signals

• Disrupts normal cellular physiology and pathways

• Leads to abnormal cellular function and disease

• Mutations that cause disease are classed as pathogenic, Mutations may have no effect are classed as benign

• Mutation and genetic variation however are drivers of evolution

Mutations in disease

Genome instability and the accumulation of mutations in even one cell can lead to disease.

Some diseases can be caused by a single nucleotide mutation.

• E.g. Sickle cell disease

  • Single A>T change

Some require multiple mutations.

• E.g. Cancer (Leukaemia)

Mutations can make cells more susceptible to genomic instability.

• E.g mutation in a DNA repair gene..leads therefore to more mutations

Q. What causes DNA damage and mutations?

Q. Damage type and extent differs by cell type – why?

Sources of genome instability

• Name 8 Exogenous sources (From outside the cell) of genome instability.

• Non-ionising radiation (UV)

• Ionising radiation (X-ray, gamma rays etc)

• Thermal damage (burns)

• Alkylating agents (tobacco smoke, chemicals)

• Chemotherapy drugs (Cisplatin)

• Viruses (Influenza virus A2/HK/68)

• Plant/fungal toxins (Aflatoxins)

• Excess hormones (Oestrogen HRT)

Sources of genome instability

• Name 7 Endogenous sources (From inside the cell) of genome instability.

• Replication errors (Fork collapse, metaphase issues, synthesis mistakes)

• Complex DNA structures (Hairpins/repetitive sequences/ RNA hybrids)

• Reactive oxygen species(metabolism products)

• Depurination (loss of A and G bases)

• Deamination (C to U conversion)

• Telomere shortening (End replication problem)

• Deficient DNA damage response (incorrect DNA repair)

Radiation is all around us

Non-ionising radiation

Ultraviolet radiation

• Normal UV (UVA and UVB) exposure can cause 100,000 DNA damage events per day

• Is the primary cause of skin cancer

• UV radiation can form a covalent link between neighbouring thymines

• Forms thymine dimers

• Interferes with DNA replication and transcription

• Usually quickly repaired by base or nucleotide excision repair

Ionising radiation

• Ionising radiation is energy released from the disintegration of atoms

• Travels as waves (gamma or X-rays)

• Or as particles (Alpha, beta or neutrons)

• Differ greatly in their energy, range of travel and ability to penetrate materials

Gamma rays vs X-rays

• What energy are they both made of?

• What can gamma rays easily penetrate?

• What can gamma rays completely pass through?

• How damaging are gamma rays?

• How do X-Rays compare in energy to gamma rays?

• ?

• ?

• What scan also uses X-Rays?

• Both made of pure energy (photons)

Gamma Rays

• Easily penetrate skin and clothing

• Can completely pass through the body

• Extremely damaging!

X-Rays

• Lower energy than gamma rays

• Can include DNA breaks

• Cells repair most of this

• CT scans also use X-rays

Ionising radiation

• What can ionising radiation directly damage?

• What are the 2 examples of this damage?

• How does radiation do this?

• What does it cause?

• What can ionising radiation generate?

• What is the example of what ionising radiation can generate?

• How does it generate this product?

• How does this product damage DNA?

• How serious is ionising radiation?

Ionising radiation can directly damage DNA

• Double strand breaks (DSBs)

• Single strand breaks (SSBs)

  • Energy from radiation breaks covalent bonds

  • A loss of DNA bases

Or generate products that are able to damage DNA

• Generate reactive oxygen species (ROS)

  • Through hydrolysis of water

  • Reactive particles attack DNA

Very serious effects and difficult to repair.

Environmental ionising radiation exposure

On average people are exposed to about 2.7 millisieverts (mSv) of ionising radiation per year (e.g in x-rays, flights, brazil nuts, e.t.c).

• Radon Gas

  • Formed from uranium found in rocks and released from the ground

  • Can be alpha, beta or gamma particles

  • UK average dose is 1.3mSv

  • (Cornwall average dose is 6.9mSv)

  • ~1000 lung cancer deaths nationally attributed to Radon

  • Homes may needed to be protected from the ground

Dont panic…

• Average = 2.7 mSv of radiation per year

• Annual limit for nuclear industry employees = 20mSv

• Level where white cell changes can be observed = 100mSv

• Radiation sickness (<50% white blood cell count) = 1000mSv

• Dose required to kill 50% of people exposed = 5000mSv

• Hisashi Ouchi - received 17 sieverts (17,000mSv)

• Radiation in research

  • We use radiation a lot in research to cause DNA damage, mutations and understand cancer

  • 32P-ATP, 3H

  • Tightly controlled

Chemically induced DNA damage

• Name 5 examples of chemicals which can induce DNA damage.

• Asbestos - can lead to oxidative DNA damage

• Pesticides - organophosphates and organochlorines (ROS)

• Mycotoxins - can crosslink DNA or form adducts (bind to DNA)

• Tobacco smoke - can crosslink DNA or form adducts (bind to DNA) + contain ROS

• Chemotherapy drugs - e.g Cisplatin crosslinks purine bases to form intrastrand adducts. Disrupts DNA structure and interferes with DNA metabolism

Endogenous sources