Protecting the Genome Flashcards
(36 cards)
Outline the causes of DNA damage
Endogenous Chemical - ROS, Spontaneous hydrolysis and Alkylation.
Exogenous Chemical - Diet (nitrosamines), Pollutants, Drugs (chemo).
Endogenous Physical - Mechanical DNA damage. Exogenous Physical - IR and UV-B.
Endogenous Biological - DNA replication errors, Transposons, Chromosome Missegregration.
Exogenous Biological - Retroviruses
Explain how ROS can damage the DNA and where they come from
ROS come from incomplete reduction of oxygen in oxidative phosphorylation. They react with the DNA and causes chemical changes to it
Explain the effect of different types of radiation on DNA
DNA absorbs 260nm most efficiently but that’s absorbed by atmosphere. UVB (295-320nm) gets through and can damage cells. It can either directly effect the DNA, or create an OH ion after interaction with water which interacts with DNA. Also, ionising radiation such as X rays and Gamma rays can damage DNA.
How can we avoid damage to our genome?
We cannot avoid ionising radiation, but we can avoid tobacco smoke, certain foods in our diet etc. Much endogenous damage is unpreventable and we must deal with it when it happens (repair).
Explain the body’s mechanism for neutralising ROS
Superoxide Dismutase enzyme catalyses the breakdown of superoxide (O2 free radicals) into Oxygen and H2O2. Catalase or Glutathione Peroxidase converts H2O2 into H2O and O2. Glutathione Peroxidase does this by oxidising Glutathione to form dimer GSSG which is reduced back to form Glutathione by Glutathione Reductase. High GSSG:GSH ratio indicates high oxidative stress.
Explain the body’s mechanism for avoiding DNA replication errors
The DNA Polymerases of the replisome have inbuilt proof reading activity, so during chain elongation they can sense misincorporation, will delete a few nucleotides using its 3’-5’ exonuclease activity, and start again.
Explain the body’s mechanism for avoiding chromosome segregation errors
The Spindle Checkpoint Assembly prevents abberant segregation. Specialised proteins in the kinetochore (centromeric protein complex) sense spindle tension - a low level will indicate that the spindles are not properly attached to the centromere, and a signal is sent which inactivates the APC/C. The APC/C triggers anaphase by the degredation of cohesin.
Describe the cause, consequence, and repair method of base modification/loss
Cause: Oxidation, Hydrolysis, Alyklation
Consequences: Point mutation, replication stalling
Repair: Base excision repair
Describe the different types of base modification
Oxidation of Guanine to 8-OxoGuanine by ROS, 8-OG will bond with A if not repaired
Depurination - Hydrolysis of G, leading to an abasic site which blocks replication and transcription
Hydrolytic deamination of C to U - U in DNA blocks replication
Hydrolytic deamination of 5-mC to T - Becomes a fixed mutation in DNA
Describe the cause, consequence, and repair method of single strand breaks
Cause: ROS, IR
Consequences: Converted to DSB by replication
Repair: HR or NHEJ (when DSB), BER
Describe the cause, consequence, and repair method of helix distorting damage like thymine dimers or Intra-strand links
Cause: UV light (T-dimers), Bifunctional alkylating agents (intralinked strands)
Consequences: Replication stalling
Repair: Nucleotide excision repair
Describe the cause, consequence, and repair method of mismatches and IDLs
Cause: Replication errors (frequency of 1/10^8) such as replication slippage or nucleotide misincorporation
Consequences: Replication stalling
Repair: Mismatch repair
Describe the cause, consequence, and repair method of Inter-strand links
Cause: Bifunctional alkylating agents
Consequence: Replication stalling, Cell death
Repair: Homologous Repair
Describe the cause, consequence, and repair method of DSBs
Cause: Replicated SSBs, ROS, IR, Mechanical Breaks
Consequences: Chromosomal deletions, inversions, translocations
Repair; NHEJ and HR (When sister chromatid available)
Describe the process of Base Excision Repair
DNA glycosylases (variable to detect different base damage) identify and remove the damaged base by cleaving the base-backbone bond to leave an AP site
A 5’ AP endonuclease cuts the sugar phosphate backbone
The baseless sugar phosphate is removed by dRase
The gap is filled and sealed by DNA Polymerase and Ligase, using the undamaged strand as a template
These last 2 steps contribute to PARP recognised ssb repair
Describe the process of Nucleotide Excision Repair
There are 2 pathways for NER, if the damage is in a region transcribed by RNA Pol II it’s Transcription Coupled Pathway, if not it’s Global Pathway. In the Global pathway, XPC recognises the damage, in TCR it’s RNA Pol II.
The DNA is unwound around the damage by helicases XPB and XPD.
XPF and XPG endonucleases make incisions a few nucleotides upstream and downstream of the damage.
25-30 nucleotides are removed, and DNA Polymerase and DNA Ligase fill and seal
Describe some diseases associated with mutations in proteins involved in NER
Xeroderma Pigmentosum is an autosomal recessive disease which is characterised by mutations in XP protein genes which causes UV light sensitivity, leading to corneal ulcers and skin dryness. Cockayne’s Syndrome is an autosomal recessive disease associate with mutations in CSA or CSB proteins which are important for post transcriptional modifications of RNA Pol II. Symptoms include short stature and neurological dysfunction and mild skin sensitivity.
Describe the process of Mismatch Repair
MutSa or MutLa recognises the mismatch/IDL.
They diffuse away from the mismatch until they reach nicks in the newly synthesised strand (this is how they differentiate new strands from parental) bound by PCNA (DNA Polymerase clamp) or RCF (loading factor).
EXO1 (exonuclease) degrades the new strand towards and through the mismatch region.
PCNA recruits DNA Pol to fill, and DNA Ligase ligates.
Hereditary Nonpolyposis Colorectal Cancer (5-7% all colon cancers) is an autosomal dominant disease which arises from mutations in any MMR related genes like MutS or MutL
Describe the process of Homologous Repair
After a dsb occurs, exonucleases bind to the ends of the exposed DNA and cleave nucleotides to give ss overhangs
RPA protects the SS overhangs
BRCA1 and BRCA2 help RAD51 to bind to the DNA, replacing RPA
The DNA end with RAD51 invades the homologous DNA, causing a Holliday Junction, and DNA Polymerase repairs the strand by using the other strand as a template
More proteins (like BLM) remove the HJ so that the two strands can seperate. DNA Polymerase and Ligase
In which other scenarios is HR important?
In meiotic division where it introduces diversity by crossing over
What happens when key HR proteins are mutated and how does it relate to cancer?
When key HR proteins like BRCA 1 and 2 have mutations, there is a decreased viability of cultured human cells. Also, mutations in BRCA1 and 2 genes are a characteristic of some cancers
What is a limitation of the fact that HR uses a template and how does the body overcome this?
If HR uses an inappropriate sequence as a template, such as a repeat region, it is genome destabilising. The body minimises this risk by inactivating the HR pathway when there is no sister chromatid present to act as a template - G1
Describe the process of Non-Homologous End Joining
Ku70/80 heterodimers bind to each end of the damaged DNA.
If the ends are blunt and undamaged they can be ligated immediately
Ku recruits the catalytic subunit of DNA protein kinase to form an active DNA-PK complex
DNA-PK recruits and activates Artemis exonuclease to trim the DNA ends and DNA Polymerase to add nucleotides
DNA Ligase is recruited to seal nicks
The end result of NHEJ is either a repaired region or an indel
What other applications does NHEJ have aside from repairing DSBs?
Used in Immunoglobulin rearrangement and T cell receptor rearrangment so deficiencies or mutations in NHEJ proteins can lead to immunodeficiency
