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Molecular Basis of Carcinogenesis II, DNA with damaged base - Coggle…

- - - - Enzyme cannot be re-used
  - - - Specific proteins
    - - Shared proteins
    - - DNA repair enzyme
      - Catalytic cysteine in active site
      - Repairs O6-methylated guanine
        
        CH\(_3\) on O6 site
        
        Mutagenic lesion (mis-pair with thymine)
      - MGMT recognises methylated guanine
        
        Protein-DNA complex formed
        
        Base flipped into active site - methyl group transferred to MGMT
        
        Repaired guanine
        
        Methylated cysteine in MGMT
      - In cancer
        
        MGMT null mice
        
        Extremely sensitive to cytotoxic + carcinogenic effects of alkylating agents
        
        ~50% of sporadic human colorectal cancer MGMT expression is lost
        
        Loss of MGMT gene
        
        Inactivating promoter hypermethylation of MGMT gene
        
        Epigenetic silencing
        
        Methylation of CpG islands (promoter)
        
        Blocks txn factor (TF) binding
        
        Decreased histone acetylation
        
        Histone H3 K9 methylation
        
        Tightly packed chromatin (heterochromatin)
    - - Base ER
        
        Non-bulky/simple forms of DNA damage
        
        e.g. simple base adducts
        
        80% cases only affected base is removed
        
        20% affected base + other unaffected bases removed
        
        Repairs damage by
        
        ROS
        
        IR
        
        Alkylation
        
        Deamination
        
        Spontaneous depurination + depyrimidination
        
        SS breaks
        
        Mechanism
        
        Simple DNA damage
        
        Removal of damaged base
        
        Cleavage of DNA backbone (SS break)
        
        DNA synthesis of correct base + ligation
        
        1 more item...
        
        Short patch repair
        
        ~80% BER activity is short patch repair
        
        Pathway varies depending upon initial glycosylase used
        
        Monofunctional glycosylases remove damaged base but leave DNA sugar-phosphate backbone intact
        
        APE1 cleaves backbone
        
        3'-OH and 5'-dRP
        
        Bifunctional glycosylases remove damaged base + cut DNA backbone
        
        e.g. Nei-like (NEIL) glycosylases
        
        Leaves 3' and 5' phosphates
        
        Leaving blocked 3'-end and 5' phosphate
        
        After DNA ends are processed, new base inserted + DNA ligated
        
        11 glycosylases recognise DNA damage
      - Nucleotide ER
        
        More bulky forms of DNA damage + some crosslinks
        
        Removes up to 30 bases around a damaged site
        
        Repairs damage by
        
        UV
        
        Bulky chemical addicts e.g. PAH from tobacco smoke
        
        Similar to BER, diff proteins involved
        
        Controlled by 'XP' family of proteins (Xeroderma Pigmentosa)
        
        Divided into global genomic repair (GGR) and transcription coupled repair (TCR)
        
        Differ in lesion recognition step
        
        GG-NER
        
        XPC-RAD23B recognises lesions that distort DNA helix
        
        TC-NER
        
        RNA Pol II is stalled during transcription to initiate NER
        
        Basic mechanism
        
        Recognition of DNA lesion
        
        DNA unwinding
        
        Dual DNA incisions
        
        Nucleotide removal, DNA synthesis and ligation
        
        After recognition, GG- and TC-NER share common pathway
        
        TFIIH recruited + unwinds DNA (XPB + XPD have helicase activity)
        
        RPA and XPA recruited to SS DNA
        
        RPA prevents strand reannealing
        
        Pre-incision complex
        
        1 more item...
        
        Defects
        
        XP is a genetic condition (autosomal recessive) caused by mutations in XP genes
        
        Leads to faulty XP proteins and defective NER
        
        Extreme sensitvity to UV light
        
        Sunburn and skin cancers
        
        Increased incidence of internal neoplasms
      - Mismatch repair
        
        Mismatched bases during DNA replication
        
        Resulting from tautomerism or triplet expansions
        
        Basic mechanism
        
        Wrong base in newly-synthesised DNA strand
        
        Recognition of base mis-match
        
        Excision of DNA segment
        
        DNA synthesis + ligation
        
        Process characterised in E.coli genes
        
        Post-replicative repair
        
        Controlled by family of 8 genes in humans
        
        Complex + not fully understood
        
        Human components named based on homology to E.coli genes
        
        MMR proteins act as heterodimers
        
        Mutations in MMR genes associated with hereditary non-polyposis colon cancer (HNPCC)
        
        Detailed mechanism
        
        Identify strand with incorrect base
        
        In E.coli, this is done by DNA methylation
        
        Mechanism unclear in other organisms
        
        Model for Exo1-dependent MMR
        
        MutS\(\alpha\) recognises DNA mismatch
        
        Binding of ATP causes MutS\(\alpha\) to slide along DNA (3'-5')
        
        Inactive Exo1 binds to DNA nicks
        
        1 more item...
        
        Nick in newly synthesised DNA strand - why?
      - Mechanisms for ER are all similar, it is size of excised SS region that varies
  - - - Detected by DNA polymerase
        
        3'-5' exonuclease activity used to remove incorrectly-incorporated base
    - - Occurs in event DNA damage is not repaired
      - Allows replication to proceed beyond damaged base
      - Requires switching of DNA polymerase
      - TLS polymerases insert a base opposite damaged base (often incorrect)
      - After lesion bypass, Pol switches back
      - Causes point mutations
        
        Relevant to large increase in point mutations seen in cancer cell genomes compared to normal tissue
  - - - MRN recruits ATM (ataxia telangiectasia mutated)
        
        ATM binds to NBS1
        
        ATM dissociates into active monomers (intermolecular phosphorylation on Serine 1981)
        
        Activated ATM phosphorylates multiple targets to coordinate DS break response
        
        ATM phosphorylates histone H2AX on Serine 139 to form \(\gamma\)-H2AX
        
        \(\gamma\)-H2AX recruits MDC1 (mediator of damage checkpoint 1)
        
        2 more items...
    - - Split into 2 pathways
        
        Non-homologous end joining
        
        Classical NHEJ involves direct ligation of DNA ends at a DS break
        
        Occurs at any stage of cell cycle
        
        DS break may not be 'clean'
        
        Small overhangs
        
        Ends are processed and directly ligated
        
        Can result in sequence errors
        
        Mechanism
        
        Binding of KU70/KU80 heterodimer to either end of DS break
        
        KU80 recruits DNA-PK\(_{CS}\)
        
        DNA-PK\(_{CS}\) = DNA dependent protein kinase catalytic subunit
        
        DNA-PK\(_{CS}\) tethers DNA ends
        
        1 more item...
        
        KU70/80 dimer is basket-shaped, DNA end sitting in central cavity
        
        Homologous recombination
        
        Requires presence of a sister chromatid
        
        Only occurs in S or G2-phases of cell cycle
        
        Sister chromatid is used as a template
        
        Error-free
        
        Requires resection of 5' ends at the DS break, forming 3' SS overhangs
        
        Crucial factor in DS break pathway choice
        
        KU70/80 cannot bind (NHEJ cannot occur)
        
        Mechanism
        
        Recruitment of MRN complex, ATM + CtIP to DS break
        
        CtIP recruitment aided by BRCA1
        
        5' end resection is initiated by CtIP and MRE11 endonucleases
        
        1 more item...
        
        Actually several sub-pathways
        
        Choice of pathway depends on cell cycle phase
        
        DS break 'condition' can also influence pathway choice
      - Defects
        
        Bloom syndrome
        
        Mutations in BLM helicase
        
        Ataxia Telangiectasia
        
        Mutations in ATM kinase
        
        Nijmegen breakage syndrome
        
        Mutations in NBS1
        
        Familial breast cancer
        
        Mutations in BRCA 1 + 2
        
        All cause genomic instability + cancer predisposition
        
        Can cause chromosome translocations
        
        Linked to some cancers e.g. Burkitt's Lymphoma
- - - - Oxidative DNA damage causes mutagenic lesion 8-oxoguanine (8-Oxo-G, mispairs with Adenine)
      - Unrepaired, causes G:C to T:A transversions
      - MUTYH = Adenine-DNA glycosylase that excises Adenine when it is wrongly paired with 8-Oxo-G
- - - - DNA pol\(\beta\) removes 5'-dRP, 5'-phosphate
        
        3'-OH and 5'-phosphate
        
        DNA pol\(\beta\) adds new base
        
        Sealing of phosphate backbone by XRCC1-DNA ligase III complex
        
        If DNA pol\(\beta\) cannot remove dRP, pathway converts to long patch repair
        
        Pol\(\beta\) switched for Pol\(\delta/\varepsilon\), synthesises a new stretch of nucelotides + displaces a flap structure
        
        Flap removed via Flap Endonuclease 1 (FEN1) + proliferating cell nuclear antigen (PCNA)
- - - - 3'-OH and 5'-phosphate
        
        DNA pol\(\beta\) adds new base
        
        Sealing of phosphate backbone by XRCC1-DNA ligase III complex
- - - - 3'-OH and 5'-phosphate
        
        DNA pol\(\beta\) adds new base
        
        Sealing of phosphate backbone by XRCC1-DNA ligase III complex