DNA damage, mutation and repair

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Created by
Zellyn Colaco

Cards (218)

  • DNA

    Chemically reactive macromolecule
  • Potential sites of modification/damage to DNA bases
    • -
    • -
  • Sources of DNA damage

    1. Hydrolysis of the amine group = deamination (doesn't occur in thymine)
    2. Forms unnatural DNA bases
    3. A > hypoxanthine (can basepair with cytosine)
    4. G > xanthine
    5. C > U (can basepair with A)
    6. Failure to repair results in DNA mutation
    7. Hydrolysis of the base and sugar-phosphate backbone
    8. Failure to repair results in deletions
  • External sources of DNA damage

    • Chemical changes in DNA from environmental mutagens
    • UV radiation
    • Chemical mutagens
  • UV radiation

    • Causes thymine dimers > causes a kink in the DNA that blocks the progression DNA polymerase
    • Failure to repair will cause the polymerase to skip over the thymine dimers > deletion
  • Chemical mutagens

    • Cause modification of the bases
    • Often small adducts (like methyl groups) but bulky adducts are possible
    • Many are polycyclic compounds that intercalate into the stacked bases of the double helix
  • Chemical mutagens

    • Benzopyrene
    • Aflatoxin
  • Aflatoxin

    • Produced by fungus - Aspergillus
    • Modified by CYP450 into active form - a DNA-modifying agent
  • Long term effects of unrepaired DNA damage
    • Mutations: alterations in the normal DNA sequence
    • Point - SNVs
    • Deletions/insertions - small to large changes (rearrangement)
  • Causes of mutations

    • Spontaneous:
    • Errors in DNA replication, proofreading
    • Tautomers - structural isomers of DNA bases
    • Deamination or loss of base
    • Frameshift - resulting from deletions or additions
    • Induced:
    • Chemicals - alkylating, intercalating
    • UV or gamma irradiation
    • Oxygen radicals
    • Base analogs - bromodeoxyuracil, hypoxanthine
  • Results of mutations at the protein level

    • Silent: codon changes but still calls for same aa
    • Missense: codon change results in different aa being incorporated into protein
    • Nonsense: changes codon into a stop codon
    • Frameshift: result from insertion or deletion of nucleotides alters all aa downstream of insertion or deletion
  • Types of DNA repair

    • Direct damage reversal
    • Excision repair
    • Mismatch repair (post-replication repair)
  • Direct damage reversal

    1. Photolyase:
    2. Photoactivation
    3. De-alkylation proteins (not catalytic):
    4. Usually repaired by base excision repair
    5. Methyl transferase can be used to remove the methyl via suicide inactivation
  • Direct damage reversal

    • Highly efficient
    • Usually requires the product of one gene and only appicable to a single lesion
    • Essentially error free
  • Base excision repair

    1. Damage bases are removed as free bases
    2. Primarily responsible for removal of oxidative and alkylation damage
    3. Major pathway for repair of modified bases, uracil misincorportation, oxidative damage
    4. Most genes in pathway are essential
    5. Proposed to have an important role in aging with significance in cancer emerging
    6. Various DNA glycosylases recognise lesion and remove base at glycosidic bond, thereby producing an abasic or AP (apurinic/apyrimidinic) site by base "flipping out"
    7. Since the intra-H bond is no longer present, the nucleotide can rotate externally and "stick out", facilitating identification by the DNA repair apparatus
    8. One of several AP endonucleases incises phosphodiesterase backbone adjacent to AP site
    9. AP nucleotide removed by exonuclease/dRPase and patch refilled by DNA synthesis and ligation
    10. Genes - glycosylase, AP endonuclease, phosphodiesterase, DNA polymerase, DNA lipase
    11. Removal of differently altered bases requires an array of N-glycosylases that each recognise a different type of damage
    12. BER imitated by glycosylases is short patch
    13. BER initiated by AP sites resulting from 'spontaneous hydrolysis' or oxidative base loss is long patch
  • Nucleotide excision repair (GG and TC-NER)

    1. Damaged bases are removed as oligonucleotides
    2. Primarily responsible for removal of UV-induced damage and bulky adducts
    3. Major repair system for removing bulky DNA lesions (like pyrimidine dimers, intra strand cross links)
    4. Thought to able to identify the phosphodiester backbone conformations created by the damage
    5. Also removes - 20% of oxidative damage
    6. Deficient in human disorders
    7. Damage recognition
    8. Dual incisions bracketing the lesion to form a 12-13 nucleotide Oligocene in Pr or a 24-32 nt Oligocene in Eu
    9. Release of the excised Oligocene
    10. Repair synthesis to fill in the resulting gap
    11. Ligation
    12. Factors required for excision: XPG, TFIIH, Replication protein A
    13. Factors required for repair synthesis: Replication Factor C, Proliferating Cell Nuclear Ag (PCNA), DNA polymerase, DNA ligase
    14. Additional factors required for transcription coupled repair: CSA, CSB
    15. Global genomic repair (GGR): Repairs all regions of the genome, Repairs all types of bulky adducts, Requires XPC and all other NER factors except CSA and CSB
    16. Transcription-Coupled repair (TCR): Repair of template strand during transcription by Pol II, Faster repair in human cells, Dependent in type of lesion e.g. cyclobutane dimers but not 6-4 photoproducts, Requires CSA, CSB and all other NER factors except XPC
  • Mismatch repair (post-replication repair)

    1. Specialised excision system which targets newly synthesised DNA strand after replication
    2. Despite extraordinary fidelity of DNA synthesis, errors do occur
    3. Such errors can be detected and repaired by the post-replication mismatch repair system
    4. Pr and Eu use a similar mechanism with common structural features
    5. Defects in MMR elevate spontaneous mutation rates 10-1000x and underlies human predisposition to colon and other cancers
    6. Primarily from DNA replication errors including template slippage - I/D loops
    7. Heteroduplex formation between homologous DNA molecules during recombination: MMR prevents homologous recombination. Defects cause micro satellite instability
    8. Changes the base in the newly synthesised strand so that it pairs with the template strand residue
    9. E.coli: Key to strand recognition is methylation of A in the GATC sites by the dam methylase, GATC site should be 5' or 3' to the mismatch and Kbs away. Therefore, exonuclease can be 3'-5' or vice versa, MutS protein finds a mismatch and complexes with MutL. They then bind to MutH, which is already bound to a hemi-methylated sequence, MutH makes a cut in the non-methylated strand. An exonuclease begins at this cleavage site and then disgests the nonmethylated strand just beyond the base mismatch, The DNA is then synthesised according to the template strand and the result is that the fault is repaired
    10. Basis: MutS dimer (in yeast, Msh2/Msh3 or Msh2/Msh6), Can recognise all base substitutions except C:C and short frameshift loops <4 bp, Transition misparis G:T and A:C and one base loops are particularly well-recognised (these are also the most common polymerase errors)
    11. Problem of strand discrimination: In E.coli, this is accomplished by the transient lack of methylation of A in GATC motifs by the dam methylase, MutH endonuclease cleaves only unmethylated GATC sites, allowing entry on newly synthesised strand, Dam mutants are mutators and show random repair of either DNA strand, In other bacteria and in Eu, the basis of strand discrimination is not understood, although entry at nicks in discontinuously synthesised DNA has been proposed
    12. Nick-directed MMR in mammalian cells: MutSa (Msh2/6) can recognised mismatch or 1bp IDL whereas MutSb (Msh2/3) recognises 2-12bp IDL, Discrimination between parent and daughter strand is accomplished by presence of nick in daughter strands, Recognition of the nick allows complexing of MutSa/MutSb with MutLa, PCNA (the processivity clamp) is required and may couple replicative machinery to MMR - PCNA interacts with M
  • Newly synthesised strand in E.coli

    1. Transient lack of methylation of A in GATC motifs by the dam methylase
    2. MutH endonuclease cleaves only unmethylated GATC sites, allowing entry on newly synthesised strand
    3. Dam mutants are mutators and show random repair of either DNA strand
  • In other bacteria and in Eu, the basis of strand discrimination is not understood, although entry at nicks in discontinuously synthesised DNA has been proposed
  • MutSa

    Msh2/6 can recognised mismatch or 1bp IDL
  • MutSb

    Msh2/3 recognises 2-12bp IDL
  • Nick-directed MMR in mammalian cells

    1. Discrimination between parent and daughter strand is accomplished by presence of nick in daughter strands
    2. Recognition of the nick allows complexing of MutSa/MutSb with MutLa
    3. PCNA (the processivity clamp) is required and may couple replicative machinery to MMR - PCNA interacts with Msh3/6
    4. A 3' to 5' (or vice versa) exonuclease is recruited to remove the newly synthesised DNA that is wrong dependent on the direction of the strand
    5. RPA protects single-stranded DNA and prevents extensive resection by exonucleases
    6. DNA polymerase preforms re synthesis
  • Hereditary nonpolyposis colon cancer: MMR mutation in 70% of families, Population prevalence = 1:2851
  • Causation of DSBs

    • X or y irradiation and chemical mutagens that generate ROS
    • Normal result of V(D)J recombination and Ig class switching processes
    • Consequence of replication fork arrest and collapse due to endogenous or exogenous DNA damage
  • Homologous recombination

    • Moving blocks of DNA from one position within the genome to another distinct position
    • Base-pairing between homologous loci, principally occurs during meiosis but is also used to repair DNA
  • Site specific recombination

    • No homology needed, used during immune system function (VDJ and TCR recombination) and DNA repair
  • Homologous recombination repair mechanism

    1. Nucleolytic processing and nucleoprotein-filament formation on the 3' end of both strands
    2. Strand invasion is initiated by RAG51 in Eu and RecA in Pr
    3. M/R/N complex 5' to 3' resection of termini, allowing the tail of the 3' end to stick out
    4. Homology search and recruitment of RAD52 (helicase) and RAD54 (end-binding protein) to form joint molecule
    5. DNA polymerase is then used to extend the strand
    6. Once the synthesis of the new strand has reached the tail of the other strand, base pairing occurs between the 2 strands
    7. DNA polymerase and ligase then facilitates gap filling and ligation
  • Information lost from broken duplex is retrieved from a homologous duplex. If a homologous duplex is not available, gene conversion may occur
  • Non-homologous end-joining mechanism

    1. Ku70/80 dimer and binds DNA ends
    2. DNA dependent PK catalytic subunit is recruited to form the DNA complex
    3. Synapsis is achieved through microhomologies
    4. Processing of DNA ends occurs through a range of factors
    5. Xrcc4/DNA ligase IV are required for the final ligation step
    6. There is release of the NHEJ machinery
  • Non-homologous end-joining

    Error prone, used for small insertions or deletions
  • Translesion DNA synthesis mechanism

    1. Lesions can block progress of replicative polymerases
    2. Specialised polymerases can perform bypasses or translesion synthesis by inserting nucleotides opposite lesions
    3. Bypass can be either error-free or error prone depending on the lesion and the polymerase
  • Polymerase Eta

    Inserts A opposite TT dimers, low fidelity and processivity, product of the XPV gene
  • Polymerases Zeta and Rev 1

    Rev 1 inserts random bases opposite dimmer, Zeta extends bypass by a few bases, both have low fidelity and processivity
  • Replicative bypass and recombination

    1. Recombination repair but the lesion remains, buys time for other repair systems to remove DNA damage
    2. Error detected (gap by dimer)
    3. Recombination occurs, causing a region of strand A to be swapped for the same region in strand C
    4. The gap in strand A now is filled by DNA polymerase and ligase, using strand B as a template
  • Not all DNA repair proteins are directly involved in lesion removal, some are components of the checkpoint apparatus
  • Cell cycle checkpoint

    Cells stop progression due to DNA damage, extends for repair, prevents DNA synthesis with damaged DNA
  • DNA structure checkpoint

    Different checkpoint proteins respond to different types of DNA damage, cells monitor DNA integrity at all stages of the cell cycle, all DNA checkpoint systems ultimately lead to an arrest in cell cycle progression, initiation of repair, and if the problem is too bad > apoptosis
  • Signal - DNA damage

    • Mismatches (replication errors)
    • Altered bases (oxidative, chemical damage)
    • Pyrimidine dimers (UV damage)
    • SS nicks (replication errors)
    • DSBs (ionising radiation)
  • Sensor - ataxia telangiectasia mutated (ATM) / ataxia-Rad related (ATR) kinases

    PI3K bind directly to the DNA, substrates include themselves (autophosphorylation), each component of the 9-1-1 complex (PCNA complex), Nbs1 or the M-R-N complex (DSB), Claspin (adaptor), P53 (cell cycle arrest and apoptosis), BRCA1 (DNA repair, transcription..), Chk1&2 kinases (cell cycle arrest), ATM is activated by DSB (in corporation with M-R-N complex), ATR is activated by other forms of DNA damage, and stalled replication, both associated with specific cancer-prone syndromes
    1. 1-1 complex

    Rad1&9&Hus1 > Rad17, PCNA-like complex: toroidal homotrimer that encircles the DNA, Rad17 complex binds to the 9-1-1 complex, opens it up and loads it onto the DNA, all components become phosphorylated by the ATM/ATR kinases to become activated