DNA repair Flashcards
Understand that... - the development of cancer requires the acquisition of new properties by cells which require genetic change in these cells - tumor cell evolution is driven by natural (clonal) selection causing non-linear diversification. The resulting tumor cells may become genetically vastly different from how they started
Altered behaviour of cancer cells
- oncogene activation -> precancerous state, increased DNA damage
- pre-cancerous state under control by checkpoints; prevent proliferation of these cells
- loss of tumor suppressor function (linked to failure of cell cycle checkpoints) enables pre-cancerous cells to proliferate
- proliferating tumor cells evolve via mutagenesis and -> altered cell functions
Hallmarks of cancer
- sustaining proliferative signaling
- evading growth suppressors
- activating invasion and metastasis
- enabling replicative imortality
- inducing angiogenesis
- resisting cell death
Cause of hyperplasia
loss of APC
Cause of dysplasia (the presence of abnormal cells within a tissue or organ)
DNA hypomethylation (loss of the methyl group in the 5-methylcytosine nucleotide)
Cause of adenoma
- activation of K-ras (early)
- loss of 18q TSG (intermediate)
- loss of p53 leads to carcinoma
Explain the cause of mutation and cancer development
multiple rounds of genetic change that lead to a growth advantage and natural selection drives tumor evolution
Describe what is meant by dynamic tumor evolution
gives rise to populations of cells with distinct proliferative advantage
The genome of a cancer cell
- change in chromosome number
- change in diploidy
- change in structure of chromosme
- genomic instability
Describe a mutation
- a change in the structure of a nucleotide sequence in a gene
- the altered sequence or gene resulting from such change
- a change in the karyotype (chromosomal)
Define mutagenesis
the process by which DNA changes, resulting in a gene mutation
Describe, with reference to experiments run using bacteria, how we know that mutagenesis requires cellular function
- low levels of mutagenesis exist in normal bacteria to allow for growth in absence of a nutrient (e.g. arginine)
- the number of cells able to grow in absence of arginine increases if bacteria is irradiated with UV light
- in cells with defective UmuD and UmuC proteins, UV light no longer induces mutagenesis
- mutagenesis, therefore, is not spontaneous
Why is mutagenesis essential
UmuD and UmuC help protect cells against DNA damaged caused by UV radiation
Describe, with reference to experiments using coal tar, how we know that DNA damage causes cancer
- coal tar damages DNA
- DNA was isolated from the cells and reintroduced into new cells
- new cells now exhibited altered behaviour, no longer respecting boundaries and growing over one another (foci)
- foci were transplanted into nude mice (compromised immune response) and induced tumors
- if repeated using DNA from cells not treated with DNA damage, no tumors form
Describe the factors contributing to DNA damage
- External sources: UV light, ionizing radiation, chemicals (e.g. treatments/smoking)
- Internal souces: ROS, depurination from metabolism, replication
Describe how mispair of DNA sequence can result in a mutation
- two adjacent nucleotides link to eachother via either cyclobutene pyrimidine dimer or 6-4 photoproduct
- linked nucleotides can no longer code, so a different replacement base enters the sequence
- complementarity is disrupted
Define chromosomal translocation
occurs when chromosome breaks and the fragmented pieces re-attach to different chromosomes, resulting in an unusual rearrangement
How does depurination / depyrimidination affect DNA sequencing
- loss of base, loss of information
- leaves non-coding lesion, which can result in changes in coding after DNA replication
The consequence of the deamination of bases
- lead to generation of non-coding bases that cannot pair with normal bases during replication
- result in coding changes after DNA replication
Reactive oxygen species (ROS)
- produced by energy production in the mitochondria
- cause DNA breaks and oxidised bases
Oxidation of guanine to 8-oxo dG
causes mispairing during replication and therefore to genetic change
Methylation of bases
mispairing of bases during replication, leading to genetic change
Exogenous agents that cause DNA damage
- UV light
- ionizing radiation
- cigarette smoke
- chemotherapy
Endogenous agents that cause DNA damage
- depurination
- deamination
- ROS
- replication errors
How are mispairs that aren’t corrected by proof reading recognised (MMR)
- mismatch repair pathway
- MutSH identifies a mismatch and slides along DNA until it reaches a nick
- helps recruit Exo1 nuclease which digests one strand of DNA from the nick to the mispair onto which loads single strand binding protein RPA
- MutSH dissociates and Polymerase delta/epsilon synthesises complementary DNA to fill the gap and remove mismatch
Base excision repair (BER)
- removal of damaged base by DNA glycosylase-sugar phosphate remains
- sugar phosphate removed by AP endonuclease and dRP lyase
- gap filled by polymerase and DNA ligase
Nucleotide excision repair (NER)
- pyrimidine dimer - DNA damage recognised
- helicases unwind DNA around damage and nucleases cut section of damaged strand 5’ and 3’ to damage
- TFIIH includes helicases XPB and XPS
- DNA polymerase and DNA ligase refill gap
Non-Homologous End Joining (NHEJ)
- loss of nucleotides near break site (resection -> leads to ss overhangs)
- Ku70/80 heterodimers are recruited, which recruit DNA-PKcs
- DNA-PKcs recruits Artemis, which gets phosphorylated and can trim the ends of the overhangs
- XLF stimulates Ligase IV to fix DSB
Alt-NHEJ (microhomology mediated End Joining)
- when Ku70/80 is not available
- ends undergo DNA resection by CtlP and MRN
- complementary sequences pair to tether broken ends (microhomology)
- Ligase joins
- can lead to extensive deletions
Homologous recombination
- occurs when you have two DNA molecules with almost the same sequence
- can occur anywhere within that length
Major pathways for repairing DNA damage
1) proofreading by DNA Polymerase during DNA replication
2) mismatch repair
3) BER
4) NER
5) break repair by NHEJ (error prone) and Alt-NHEJ
6) break repair by homologous recombination (error free)
Why is it important to fix DSB
- can block replication fork
- can lead to loss of chromosome segment
- can lead to apoptosis
How can DSB be fixed
- homologous recombination (if damage occurs in G2/M phase where a good copy of DNA is available)
- NHEJ (if damage occurs in G1/S phase where there is no copy to use as a template)
Why is homologous recombination favoured over NHEJ?
- NHEJ is error prone
- can lead to loss/gain of genetic information
Role of RAG1 and RAG2 proteins
- end of VDJ genes
- shuffle, separate, and rejoin the VDJ genes
- shuffling takes place inside B and T cells during maturation
Role of NHEJ in antibody diversity
takes place in VDJ recombination which creates antibody diversity
Define microhomology
the presence of the same short sequence of bases in different genes
