DNA damage and repair

Cards (17)

  • Describe the causes and types of DNA damage
    DNA can be damaged by Both endogenous sources, such as replication stress or metabolic reactions producing ROS, or external sources, such as radiation. This can lead to base mismatches, single stranded breaks (SSBs), intrastrand or interstrand crosslinks and double stranded breaks (DSBs). These DNA lesions occur very frequently, so it is important the cells are adapted to deal with them rapidly.
  • Describe the DNA damage response 

    DNA damage sensor proteins detect DNA lesions and recruit mediator proteins. This leads to signal amplification, which coordinates multiple processes via activation of effector proteins:
    • Halt cell cycle: prevent inheritance of mutation
    • DNA repair: specific for each DNA lesion
    • Chromatin changes: allow access to DNA for repair
    • Gene expression
    • Cell fate: induce Apoptosis or senescence
  • How does DSB repair fluctuate over the cell cycle?
    G1 phase: in this stage of the cell cycle there is no homologous chromosome, so ATM, a major upstream DDR kinase, is used to activate CHK2. CHK2 phosphorylates p53, which promotese accumulation and induces p53-mediated apoptosis.
    S phase: in this stage of the cell cycle there is homologous chromosomes, so ATR, a major upstream DDR kinase, initiates DSB repair and activates CHK1, which activates DNA damage checkpoints. This allows DNA to be repaired via homologous recombination.
  • Describe the different repair mechanisms in the cell cycle
    G1 phase: non-homologous end joining can be used to repair breaks in the DNA, however this is usually mutagenic as no homologous sequence is used.
    S phase: homologous recombination is used to repair breaks in the DNA as this is the most accurate mechanism and uses the homologous chromosome. This is regulated by BRCA1, BRCA2 and RAD51 (and many others).
  • Are DNA lesions static?

    DNA lesions are not static and can transition into different lesions, for example SSBs, if not repaired quickly by PARP enzyme, can transition to form DSBs.
  • What are PARP enzymes?

    These are poly ADP-ribose polymerases that play a crucial role in the repair of SSBs, specific PARP-1. PARP-1 is activated by DNA breaks and adds successive ADP-ribose units to form a long branched chain that covalently attaches to histone and DNA repair proteins. These units essentially form a scaffold to recruit proteins essential in SSB repair.
  • Describe how DNA damage is useful 

    Meiotic recombination: DNA break repair via homologous recombination promotes pairing of homologous paternal and maternal chromosomes. This increases genetic diversity of gametes.
    Immune system: DSB induction and repair facilitates VDJ recombination generates diversity in antibodies during B cell development. Also involved in class switching recombination.
    Neural plasticity: SSB repair modulates neural plasticity by altering synaptic transmission.
  • Describe diseases associated with defects in the DNA damage response
    Werner syndrome: caused by defects in WRN helicase, which unwinds DNA for repair.
    Bloom syndrome: caused by defects in BLM helicase, which is involved in the repair of DSBs
    Fanconi anaemia: caused by defects in the FANC genes, which encode proteins for the FA core complex. This complex is important in the repair of DSBs.
  • DNA damage in cancer can be described as a double-edge sword:
    • Benefits: increased mutational burden can increase oncogene activation and loss of tumour suppressors, leading to tumourigenesis
    • Drawbacks: genome instability leads to increased DNA damage and replication stress, which can result in cell death.
  • Describe germline vs somatic mutations in cancer cells

    Germline variants: variant is present in gamete and therefore present within every cell of the body, including the germline so it can be passed onto offspring.
    Somatic variants: variant is absent in gametes, so variation will not be in all the cells and is not passed onto offspring.
    Recessive germline mutations will be masked unless a somatic mutation occurs within the other copy of the gene.
  • BRCA deficient cancers, such as ovarian cancers, can be selectively targeted with PARP inhibitors, such as Olaparib. Selective targeting is facilitated by a mechanism called synthetic lethality, which is when mutations in two genes results in cell death, but a mutation in either gene alone does not. In this case inactivation of PARP and BRCA1/2 leads to cell death.
  • Why are PARP inhibitors synthetically lethal with BRCA deficiency?
    This is due to the cross talk between DNA repair pathways, which back each other up. Therefore, BRCA deficiency sensitises cancer cells to the action of PARP inhibitors as there is no back up for DNA repair.
  • How can we identify patients that can receive PARP inhibitor treatment via DNA sequencing?
    Biomarker tests are required for identifying BRCA1/2, and other homologous recombination genes such as PALB2, mutations to stratify patients for PARP inhibitor treatment. However, other genes may also contribute to HR deficiency and this does not take into account epigenetics inactivation of BRCA.
  • How can identify patients for PARP inhibitor treatment using functional assays?

    Collect tumour tissue and conduct immunofluorescence assay for RAD51, which is a marker for DNA damage since it is recruited to DNA lesions in order to facilitate repair via homologous recombination. This method is easy and rapid, as well as reliable.
  • Describe resistance to PARP inhibitors
    Cancer cells can partially reactivate BRCA genes via reversion mutations or promoter switching through chromosomal translocation.
  • How do we understand synthetic lethal relationships?

    Large scale lethality screens can be conducted, based on the CRISPR Cas9 system. This would improve understanding of other lethal synthetic relationships. ATM deficiency, which is common in many cancers, is synthetic lethal with inhibitors of several DNA repair enzymes, such as PARP inhibitors and DNA-PKcs inhibitors. Microsatellite instability is synthetic lethal with Werner inhibitors.
  • Why do we need to understand synthetic lethal relationships?
    Many other synthetic lethal relationships have been uncovered, but further clinical testing, developing of pharmacological inhibits and fundamental discovery work is required to optimise patient Stratification and clinical outcomes.