Genomic instabillity Flashcards
Why is genomic stability important?
Genomic stability is important for cell growth and avoidance of development of cancer and genome instability syndromes.
- A plethora of damage response mechanisms collectively act to maintain genomic stability in the face of continuous DNA damage incurred by exogenously and endogenously generated DNA damaging agents. The importance of these mechanisms is underscored by the fact that several hereditary syndromes associated with developmental abnormalities and cancer predisposition harbour mutations in essential components of these pathways, e.g. LIG4 syndrome, RS-SCID, XLF defect, XP etc.
Outline LIG4 syndrome.
LIG4 syndrome is an autosomal hereditary disorder conferred by mutations in DNA ligase IV, a protein that plays a pivotal role in DNA non-homologous end-joining (NHEJ) and V(D)J recombination. DNA ligase IV co-associates with XRCC4 and functions in the final rejoining step of NHEJ. DNA ligase IV has a conserved ligase domain at its N-terminal and two C-terminal BRCT domains. Interaction with XRCC4 occurs via the region that lies between the two BRCT domains.
As DNA ligase IV is essential for development in mice (LIG4 -/- is embryonic lethal), mutations in the LIG4 syndrome patients are hypomorphic (partial loss of gene function). Five LIG4 syndrome patients have been described:
- One individual, identified by his over-response to radiotherapy, was clinically normal until the onset of leukaemia at age 14 (R278H).
- The remaining patients displayed pancytopaenia, slow development and growth delay.
The R278H mutation impacts upon DNA ligase IV function reducing the activity to ∼10% of wild-type (WT) levels. Clinical severity among the 5 patients with LIG4 syndrome correlated with the level of residual ligase activity.
Name and outline a syndrome that arises from defective Artemis.
Artemis codes for a V(D)J recombination/DNA repair factor that belongs to the metallo-β-lactamase superfamily and whose mutations cause human RS-SCID condition.
Among the genetic defects that cause T-B− SCID are biallelic mutations in DCLRE1C, initially identified in a subset of T-B− SCID patients with increased radiosensitivity (RS-SCID). DCLRE1C encodes ARTEMIS, a nuclease with intrinsic 5′-3′ exonuclease activity on single-stranded DNA.
- After phosphorylation by and in complex with DNA-dependent protein kinase catalytic subunit, ARTEMIS acquires endonuclease activity on 5′ and 3′ overhangs, and hairpins.
- It is involved in non-homologous end-joining (NHEJ) and is essential for opening hairpins, which arise as intermediates during V(D)J recombination of the immunoglobulin and T-cell receptor genes in T- and B-cell development.
Severe combined immunodeficiency (SCID) is a rare disorder presenting in infancy with life-threatening infections (bacterial, viral or fungal), failure to thrive and diarrhea.
- SCID can be caused by mutations in various genes, predominantly affecting T-cell immunity. In SCID, T-cell activation and function are impaired, or T-cell development is hampered causing low or absent peripheral T cells. Distinct genetic forms of SCID can be subdivided into T-B+, T-B− or T+B+ SCID, depending on the presence/absence of the respective cell line.
Outline how patient 2BN lead to the discovery of a new NHEJ component.
Defects in any of the factors involved in DNA DSB repair can lead to pronounced radiosensitivity and immunodeficiency. Cells were analysed which were derived from a patient with a clinical presentation that would be consistent with such a defect (patient 2BN):
- 2BN cells are dramatically radiosensitive and defective in DNA DSB rejoining.
- 2BN cells are not defective in any of the characterized NHEJ activities: Western blotting with specific antibodies to Ku70, Ku80, DNA-PKcs, DNA ligase IV, and Xrcc4 demonstrated that all five proteins were expressed at normal levels. All genes had wild-type sequences. 2BN cells expressed normal levels of Mre11, Rad50, and Nbs1 by Western immunoblotting. Transient transfection of cDNAs expressing hMre11, hRad50, or Nbs1 failed to complement the V(D)J defect.
This lead to the search for the novel factor involved in DNA DSB repair by two groups: Cambridge (studied 2BN) and a French group (studied 5 new patients).
- Cambridge: performed a yeast hybrid screen for XRCC4-interacting protein (expected to structural similarity). A G>T mutation was found in exon 11 of the XLF gene, and reintroduction of WT XLF rescued radiosensitivity.
- The French group: genetic analysis using highly polymorphic microsatellite markers allowed them to formally exclude a role for the six known NHEJ factor-encoding genes Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, and Artemis. They designed a functional complementation cloning strategy based on the rescue of the increased cellular sensitivity to DNA-damaging agents to identify the defective gene. cDNA library was introduced into patient’s cells and treated with bleomycin, complementation was seen in some cells and the cDNA was identified. Named new protein Cernunnos.
What are some sources of DNA damage?
Radiation:
-
Ionising
- alpha and beta particles
- X-rays and gamma rays
-
Non-ionising
- UV radiation
Endogenous agents:
- Reactive oxygen species (ROS)
- Replication
Chemical agents:
- Alkylating agents
- Acridine
How is DDR arranged heirarchicaly?
- Sensors
- Mediators
- Transducers (ATM/ATR)
- Effectors
How does a cell create genome stability? What happens when this doesn’t work?
Cells have evolved a highly co-ordinated cellular system to sense and conteract DNA damage called the DNA damage response (DDR). This is tightly linked to the cell cycle to ensure damage is not replicated or amplified by further progression, creating genome stability. When components of DDR are defective, this leads to genome instability.
Name some genomic instability syndromes and their defective components.
- Xeroderma pigmentosum (XP): defective NER due to mutations in ERCC1-5, DDB2, XPA, XPC, and POLH. Diagnosed in complementation groups A-G.
- Ataxia telangiectasia (AT): defective DSB repair due to mutations in ATM.
- Fanconi anaemia (FA): defective HR and sometimes NHEJ due to mutations most commonly in BRCA2, BRIP1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, and less commonly in ERCC4, FANCL, FANCM, MAD2L2, PALB2, RAD51, RAD51C, SLX4, UBE2T, XRCC2.
- RS-SCID: defective NHEJ due to mutations in Artemis.
- LIG4 syndrome: defective NHEJ due to mutations in LIG4.
- XLF defect: defective NHEJ due to mutations in XLF.
HANNBAMFS
What are the different types of DDR? [8]
- Mismatch repair (MMR)
- Base excision repair (BER) and single-strand break repair (SSBR)
- Nucleotide excision repair (NER)
- Non-homologous end joining (NHEJ)
- Homologous recombination (HR)
- Fanconi anaemia (FANC) pathway
- ATM-mediated DDR signalling
- ATR-mediated DDR signalling
What lesions does BER respond to? What proteins are involved?
Abnormal DNA bases (deaminated C→U). Components vary based on whether it is short-patch or long-patch repair.
- DNA glycosylases (sensors)
- APE1 endonucleases
- DNA polymerases (β, δ, ε) and associated factors
- Ligase I or ligase III
What lesions does NHEJ respond to? What proteins are involved?
Chemically- or radiation-induced DNA DSBs, and V(D)J intermediates.
- Sensors Ku70/Ku80
- DNA-PKcs
- XRCC4
- XLF/Cernunnos
- Ligase IV
- Can also emply MRN complex and other proteins (alt-NHEJ).
What lesions does NER respond to? What proteins are involved?
Lesions that disrupt the double helix, such as bulky base adducts and UV photo-products. Two pathways: global genome repair (GGR) and transcription coupled repair (TCR).
- Sensors: elongating RNA Pol II, XPC-HR23B.
- XPA
- XPE
- XPF/ERCC1
- XPG
- CSA and CSB
- TFIIH (containing helicases XPB and XPD)
- RPA
- Ligase I
What lesions does HR respond to? What proteins are involved?
DSBs, stalled replication forks, inter-strand DNA crosslinks.
-
RAD51 and related proteins:
- XRCC2, XRCC3, RAD51B-D, DMC1
- RAD52
- RAD54
- BRCA2
- RPA
- FEN1
- DNA Pol and associated factors
- Promoted by: MRN (Mre11-Rad50-Nbs1), CtIP, BRCA1, and ATM signalling pathway.
The DRR of DDR
What are the three main steps in any DDR pathway?
- Detection of DNA damage
- Recruitment of DNA repair factors
- Repair of DNA lesions
Give some examples of DDR:
- sensors
- mediators
- transducers
- effectors
- H2AX, MRN (Mre11-Rad50-Nbs1), XPC, Ku70/Ku80
- BRCA1
- ATM, ATR, DNA-PK, Chk1, Chk2
- E2F1, p53, CDC25A
Outline the steps in NER. [6]
- Damage recognition by XPC or DNA Pol II
- DNA is unwound ~20 bp around the damaged site by the helicase activity of TFIIH (XPB and XPD)
- XPA, RPA, XPG recruited and XPC is released
- XPG and ERCC1-XPF associate with TFIIH
- ERCC1-XPF makes the first incision and XPG incises the 3’ single/double strand junction
- The damaged strand is displaced , DNA Polymerase and associated proteins fill the gap, and DNA Ligase I seals the gap
What is the function of ATM? What are the consequences when it is dysfuncional?
- Normally: ATM belongs to a conserved serine/threonine kinase family (PIKKs) that all contain a PI3K domain. This is the catalytic site of the active protein. ATM responds primarily to DSBs. ATM resides inactive in the nucleus in a dimer, where each PI3K domain is blocked by the FAT domain on the other molecule. Following DNA damage, each ATM molecule phosphorylates the other on Ser1981 in the FAT domain, releasing the molecules into active monomers. ATM then proceeds to phosphorylate a wide array of substrates involved in DDR.
- Dyfunctionally: ataxia telangiectasia is the genomic instability syndrome resulting from a mutant ATM gene. It is characterised by cerebellar degeneration, leading to severe neuromotor dysfunction, immunodeficiency, genome instability, thymic atrophy, extreme IR sensitivity, predisposition to lymphoreticular malignancies.
