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BB3701 Genomic medicine > Genomic instabillity > Flashcards

Flashcards in Genomic instabillity Deck (24)
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1
Q

Why is genomic stability important?

A

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.
2
Q

Outline LIG4 syndrome.

A

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:

  1. One individual, identified by his over-response to radiotherapy, was clinically normal until the onset of leukaemia at age 14 (R278H).
  2. 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.

3
Q

Name and outline a syndrome that arises from defective Artemis.

A

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.
4
Q

Outline how patient 2BN lead to the discovery of a new NHEJ component.

A

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.
5
Q

What are some sources of DNA damage?

A

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
6
Q

How is DDR arranged heirarchicaly?

A
  1. Sensors
  2. Mediators
  3. Transducers (ATM/ATR)
  4. Effectors
7
Q

How does a cell create genome stability? What happens when this doesn’t work?

A

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.

8
Q

Name some genomic instability syndromes and their defective components.

A
  • 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.
9
Q

HANNBAMFS

What are the different types of DDR? [8]

A
  1. Mismatch repair (MMR)
  2. Base excision repair (BER) and single-strand break repair (SSBR)
  3. Nucleotide excision repair (NER)
  4. Non-homologous end joining (NHEJ)
  5. Homologous recombination (HR)
  6. Fanconi anaemia (FANC) pathway
  7. ATM-mediated DDR signalling
  8. ATR-mediated DDR signalling
10
Q

What lesions does BER respond to? What proteins are involved?

A

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
11
Q

What lesions does NHEJ respond to? What proteins are involved?

A

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).
12
Q

What lesions does NER respond to? What proteins are involved?

A

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
13
Q

What lesions does HR respond to? What proteins are involved?

A

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.
14
Q

The DRR of DDR

What are the three main steps in any DDR pathway?

A
  1. Detection of DNA damage
  2. Recruitment of DNA repair factors
  3. Repair of DNA lesions
15
Q

Give some examples of DDR:

  1. sensors
  2. mediators
  3. transducers
  4. effectors
A
  1. H2AX, MRN (Mre11-Rad50-Nbs1), XPC, Ku70/Ku80
  2. BRCA1
  3. ATM, ATR, DNA-PK, Chk1, Chk2
  4. E2F1, p53, CDC25A
16
Q

Outline the steps in NER. [6]

A
  1. Damage recognition by XPC or DNA Pol II
  2. DNA is unwound ~20 bp around the damaged site by the helicase activity of TFIIH (XPB and XPD)
  3. XPA, RPA, XPG recruited and XPC is released
  4. XPG and ERCC1-XPF associate with TFIIH
  5. ERCC1-XPF makes the first incision and XPG incises the 3’ single/double strand junction
  6. The damaged strand is displaced , DNA Polymerase and associated proteins fill the gap, and DNA Ligase I seals the gap
17
Q

What are the differences between NHEJ and HR?

A
  • NHEJ: employed mainly during G1, error-prone.
  • HR: employed onlyduring G2, error-free.
18
Q

Ahhh quick!!!

Quick, there’s a DSB in the DNA!! What does the cell do?!?

A

DSBs recruit and activate ATM which causes histone H2AX phosphorylation (γ-H2AX) on S139 at the site of DNA damage, a crucial step in the DDR. The γ-H2AX then recruits additional ATM to the site of DSBs, which causes further spreading of γ-H2AX along the chromatin, facilitated by DDR mediators such as MDC1 and p53BP1.

  • MDC1 (a regulator of the Intra-S phase and the G2/M cell cycle checkpoints that recruits repair proteins to the site of DNA damage).

The downstream DDR effectors, which function far from the site of DNA damage, are activated when the local activity of ATM/ATR increases above a certain threshold. ATM phosphorylates and activates the transducer kinase Chk2 specifically, whereas ATR phosphorylates Chk1 preferentially. Once activated, both Chk1 and Chk2 diffuse throughout the nucleus spreading DDR signalling. Ultimately, the DDR signalling pathways converge to inactivate CDC25, an essential phosphatase for cell cycle progression and to activate p53, which in turn induces p21. This, however, happens in two waves.

  1. First, there is rapid cell cycle arrest caused by destruction of CDC25 (mediated by Chk1), which is followed by induction and stabilisation of p53, which leads to stable cell cycle arres.
  2. During the DDR, γ-H2AX spreads hundreds of kilobases away from the DNA-damage site and recruits checkpoint signalling complexes and DDR factors leading to formation of DNA-damage foci.

If DNA damage is repaired, then the DNA-damage foci are disassembled as cells resume normal function.

19
Q

Outline Ku70/Ku80 function. What happens when it is dysfunctional?

A
  • Normally: Ku70/80 rapidly binds DNA DSBs, loading the broken end into its toroidal structure. It also possesses DNA processing activity. After binding DNA it loads and activates DNA-PK to initiate NHEJ.
  • Dysfunctional: there are no known human defects. In Ku-/- mice there are severe growth abnormalities, SCID phenotype, radiosensitivity, cancer predisposition, lymphoma development and premature ageing.
20
Q

Outline DNA-PK function. What happens when it is dysfunctional?

A
  • Normally: DNA-PKcs binds and is activated by Ku70/Ku80 at DNA DSBs. Following DSB binding, DNA-PKcs undergoes autophosphorylation on the T2609 cluster, providing access to end processing enzymes such as Artemis (promotes access for resecting enzymes in HR when NHEJ fails). Excessive end processing is prevented by DNA-PKcs authophosphorylation on the S2056 cluster, protecting the DNA ends (inhibiting HR by preventing end resection). Following this LIG4-XRCC4 and XLF are recruited to ligate the broken ends.
  • Dysfunctional: There were no reported cases of dysfunctional DNA-PKcs in humans until recently (2009: L306R missense mutation causing insufficient Artemis activation. Causes RS-SCID). DNA-PKcs-/- mice show growth abnormalities, SCID phenotype, premature ageing, radiosensitivity, more severe kyphosis, early lymphoma development, intestinal atrophy, and increased susceptibility to infection.
21
Q

What is the MRN complex? What are the functions of the subunits?

A

Mre11-Rad50-Nbs1 (MRN) prepares DNA for HR.

  • Rad50: contains ATPase domains that interacts with Mre11 and associates with DNA ends.
  • Mre11: stabilises DNA ends and has endonuclease and exonuclease activities needed in DNA resection during HR.
  • Nbs1: interacts with Mre11 and recruits ATM to the site of DNA damage through its c-terminal region.
22
Q

Outline Ligase IV function. What happens when it is dysfunctional?

A
  • Normal: Ligase IV is an ATP-dependent DNA ligase that joins double-strand breaks during the non-homologous end joining pathway of DSB repair. It is also essential for V(D)J recombination. Lig4 forms a complex with XRCC4, and further interacts with the DNA-dependent protein kinase (DNA-PK) and XLF/Cernunnos, which are also required for NHEJ. Ligase IV is encoded by the LIG4 found on chromosome 13q33.
  • Dyfunctional: In humans, mutant ligase 4 results in a clinical condition known as LIG4 syndrome, and autosomal hereditary disease. Mutations are thought to be hypomorphic. This syndrome is characterized by radiosensitivity, growth delay, developmental delay, microcephaly, facial dysmorphisms (‘Bird-like’), increased disposition to leukemia, impaired DSB rejoining, variable degrees of immunodeficiency and pancytopenia. Transgenic mouse models can be used to study this syndrome: R278H LIG4 mutant, defective V(D)J recombination, radiosensitivity, LIG4-/- embryonic lethal (rescued by p53), growth retardation, decreased life span, decreased fertility.
23
Q

What is the function of ATM? What are the consequences when it is dysfuncional?

A
  • 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.
24
Q

How can AT be diagnosed?

A
  • Clinical symptoms: early onset ataxia with oculocutaneous telangiectases, with oculomotor apraxia for early diagnosis (which is usually difficult).
  • Elevated alpha-fetoprotein (95% cases) and carcinoembryonic antigen.
  • Increased frequency of breaks and rearrangements in chromosomes, which can be detected under microscopy after exposing cells to IR/bleomycin.
  • ATM sequence analysis and mutation scanning.