Intro: sources and consequences of genomic instability

Question 1

How frequent are DNA lesions in cells?

Answer 1

~7 lesions per human cell per minute

Question 2

What are the problems associated with DNA breaks? (2)

Answer 2

• If cells survive it can lead to cancer because of genomic rearrangements/mutations in tumour suppressor genes/oncogenes
• Cell death by apoptosis causes degenerative diseases and autoinflammation

Question 3

When are programmed DNA breaks important? (3)

Answer 3

• Immunoglobulin diversity
• Meiotic recombination
• Antigenic variation

Question 4

What is immunoglobulin diversity?

Answer 4

Reorganisation of immunoglobulin genes by V(D)J recombination, including generation of breaks by RAG1 and RAG2 to enable antibodies to target a range of antigens

Question 5

What is meiotic recombination? (3)

Answer 5

• Recombination between homologous chromosomes is an important feature of meiosis I
• Physically holds homologous chromosomes together and generates genetic diversity
• DSBs are deliberately formed to initiate meiotic recombination involving Spo11

Question 6

What is antigenic variation? (2)

Answer 6

• Trypanosoma brucei parasite expresses variant-surface glycoproteins (VSGs) on their surface to mask invariant surface antigens
• Express one at a time and switch which VSG is expressed from a repertoire of hundreds of silent genes using homologous recombination

Question 7

What is spontaneous base loss? (3)

Answer 7

• Loss of a base due to hydrolysis because of the alkaline environment in the cell
• 1800 purines and 600 pyrimidines per cell per day
• Results in no template for replication and unstable site which can lead to a ss or ds break

Question 8

What is deamination? (3)

Answer 8

• Loss of an NH2 group from a base due to spontaneous hydrolysis/specific enzyme activity
• Cytosine is converted to uracil which is replaced by thymine in the next DNA replication and pairs with adenine, resulting in a C>T point mutation
• 5-methylcytosine (in CpG dinucleotides) are converted straight to thymine by deamination

Question 9

What is ROS? (3)

Answer 9

• Reactive oxygen species derived from a range of cellular metabolic processes
• Oxidising agents which ‘want’ electrons from other molecules
• E.g. H2O2 hydrogen peroxide, O2- superoxide, OH hydroxyl radical

Question 10

Where does ROS come from in the cell? (2)

Answer 10

• Over 50 endogenous human enzymes generating ROS, notably the mitochondrial electron transport chain
• Redox signalling affects cellular protein function by reversibly oxidising cysteine residues

Question 11

What is oxidation?

Answer 11

Loss of electrons

Question 12

What is 8-oxoguanine? (2)

Answer 12

• Oxidised G that can either pair with C (as normal) or with A
• ROS reacts with double bonds in DNA (steals electrons)

Question 13

How does histone demethylation generate ROS? (3)

Answer 13

• During the action of demethylase LSD1, first step is oxidation by FAD
• FAD is regenerated with creation of H2O2
• Similar during demethylation of DNA (5-methylcytosine)

Question 14

What is an R loop? (3)

Answer 14

• Structure that can form during transcription particularly is transcription is slow
• Newly made RNA forms a duplex with the template DNA strand, leaving the displaced ssDNA non-template strand which is vulnerable to damage
• Supercoiling on either side of the R loop

Question 15

What problems are caused by R loops? (2)

Answer 15

• ssDNA is vulnerable to ROS and inappropriate enzymatic processing
• Can cause replication fork stalling due to DNA polymerase colliding with R loop, ssDNA can form secondary structures, supercoiling etc leading to DNA breaks

Question 16

What is ribose contamination? (3)

Answer 16

• When ribonucleotides get incorporated into the DNA duplex during replication instead of deoxyribonucleotides
• Can be due to incomplete removal of Okazaki fragments/mistake by DNA polymerase because 100X more ribonucleotides present in the cell as they need to be constantly available for transcription
• Problem because the OH on ribose makes it much more reactive than the deoxy version

Question 17

What are topoisomerases? (2)

Answer 17

• Enzymes which cut DNA and then re-seal to relieve the stress on the duplex due to supercoiling generated by transcription/replication
• Topoisomerase is covalently bound during this process so if the reaction can’t be completed the DNA-protein complex becomes trapped and needs to be fixed