Faithful DNA replication health - individual survival - species
Species Mutations in somatic cells
DNA MUTATIONS and
in germline cells Evolution
established after 2 cell divisions
REPAIR
1 stable change per average protein in 200 000 years
Cell changes: cell death / disease / survival & division
new species
mostly deleterious for the affected individual
Somatic cells must be protected to safeguard individuals
Mutations occur continuously at a low rate - difficult to estimate Rate / cell cycle (in tissue culture) = 1 mutation / 109 b.p. (7x109 b.p./ human genome)
DNA repair mechanisms
Mutation established after 2 DNA replications (cell divisions)
Accurate DNA replication
DNA Change
Repair mechanism unavailable / faulty
Cause
Missing base
Depurination
Incorrect base
Spontaneous deamination Alkylating agents Tautomeric shifts Ionizing radiation Replication Mismatch
Repair Base Excision Repair
Deletion/Insertion Intercalating agents Strand breaks
Ionizing radiation Chemicals
WHAT CAUSES MUTATIONS? SPONTANEOUS Extensive damage occurs continuously. continuously Faulty ‘etidoryal’ proofreading during replication. CHEMICAL Chemicals from the environment (mutagens, carcinogens) (some activation by our own metabolism) Modification of bases (alkylation) Insertion between bases UV, IONIZING RADIATION Cross linking of base pairs Ring opening DNA strand breaks
DNA Change
Cause
Missing base
Depurination (104/day/cell)
Incorrect base
Spontaneous deamination Alkylating agents Tautomeric shifts Ionizing radiation Replication Mismatch
Thymine dimers
UV radiation
Deletion / insertion
Intercalating agents
Strand breaks
Ionizing radiation Chemicals
Mismatch Repair
Thymine dimers UV radiation Nucleotide Excision Repair Photolyase
essential for longterm survival of life
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DEPURINATION (104 / cell / day)
Missing base:
Incorrect base: SPONTANEOUS DEAMINATION (102 / cell / day) e.g. C deaminates to U
Spontaneously and through heat/acid
NH2
Spontaneous depurination
- base is lost - sugar-phosphate backbone is left
- if not repaired, strand cannot replicate
N
Deamination
O
Normal base G C
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2
Deamination C→ →U
GC
GC
AT
3
Cause
Missing base
Depurination (104/day/cell)
Incorrect base
Spontaneous deamination Alkylating agents Tautomeric shifts Distort DNA: Ionizing radiation Uvr-ABC Replication Mismatch Nucleotide UV radiation Excision Repair Intercalating agents
Strand breaks
Ionizing radiation → Bulky lesions Chemicals
O
Cytosine
Uracil
Thymine
Base excision repair Uracil DNA glycosylase
1. Recognition-removal of base Base-specific glycosylases remove incorrect base. Note: Repair of depurinated base pairs starts here.
2. Excision
AU
DNA Change
Deletion / insertion
N
Sugar-phosphate backbone is excised.
Needs to be repaired here, before the mutation passes to the next generation
Thymine dimers
O
NH
sugar
AP endo-nuclease & Phosphodiesterase
AU
N
CH3
sugar
GU
GC
NH
sugar
Deamination of C: G-C → A-T transition mutation (102/cell/day) Generation
O
O N
Polymerase I Ligase
3. Resynthesis - DNA Pol I 4. Ligation - Ligase
Incorrect base:
ALKYLATING AGENTS
Alkalytion: adds methyl or ethyl groups to O & N of bases & phosphates of the DNA strand Effects: (1) disrupts normal base pairing - strands distort (2) DNA strands break or form inter-strand bridges (3) Replication = faulty Source: environmental carcinogens / mutagens, eg Methylnitrosourea (MNU) Ethylmethanesulfonate (EMS) Chemotherapeutic agents (e.g. Cisplatin)
2
Thymine dimers:
UV RADIATION 2 adjacent thymine residues 6-4 photoproduct dimerization occurs with T or C (i.e. pyrimidines)
UV light
T-T dimer is pulled together, A’s are not hydrogen bonded Replication stops at this point
UV energy is absorbed by T
In normal cells, 50% is repaired in < 24 hours
Deletion/insertion: INTERCALATING AGENTS Stretching changes the frame needed by DNA Polymerase during replication.
Cyclobutane thymine dimer
Deletion/insertion: INTERCALATING AGENTS Intercalating agents: flat, multi-ring aromatic molecules For example:
Extra nucleotides are added during replication The DNA reading frame for RNA synthesis is changed: amino acid changes altered protein Original DNA
A A B
B C
Benz[a]anthracene from cigarette smoke & charred meat activated by our liver enzymes
Ethidium Bromide
Aflatoxin: from mouldy peanuts
(DNA stain used in Laboratory)
DNA stretched by intercalated ligands
Thymine dimer 5’
6-4 photoproduct
O
Uvr ABC Nucleotide excision repair 3’ 1. RecognitionDNA bending UvrA, UVrB ATP 2. Excision-Excinuclease UvrA dissociates UvrB~UvrC UvrD helicase unwinds fragment
Repair system = NUCLEOTIDE EXCISION REPAIR For DNA distorting lesions in prokaryotes, e.g. E.coli: Uvr ABC nucleotide excision repair e.g. alkyl adducts, UV-induced dimers of bases, intercalating agents)
In eukaryotic cells, e.g. humans,
17 proteins cooperate in nucleotide excision repair. 3. Resynthesis
Polymerase I or II 4. Ligation
Ligase
In the disease Xeroderma Pigmentosum some of these 17 components are mutated & non-functional. Investigation of XP patients helped to identify some of the components (named: XPA, XPB, XPC…XPG )
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DNA Change
Cause
Repair
Missing base
Depurination
Incorrect base
Spontaneous deamination Alkylating agents Tautomeric shifts Ionizing radiation Replication Mismatch
Photolyase - Photoreactivation
Photolyase contains two
Base Excision Repair
chromophores (can absorb light of a characteristic wavelength)
Mismatch Repair
Thymine dimers UV radiation Nucleotide Excision Repair
Deletion/Insertion Intercalating agents Strand breaks
Ionizing radiation Chemicals
Photolyase
1. THF (tetrahydrofolate) derivative harvests light energy & transfers it to 2nd chromophore 2. FADH – transfers electrons to pyrimidine dimers to break the dimerising bonds
Mismatch DNA repair
DNA Change
Cause
Missing base
Depurination (104/day/cell)
Incorrect base
Spontaneous deamination Alkylating agents Tautomeric shifts Ionizing radiation Replication Mismatch
Thymine dimers
UV radiation
Deletion / insertion
Intercalating agents
Strand breaks
Ionizing radiation → Bulky lesions Chemicals
Despite proofreading during replication SOME mismatch replication errors occur The Mismatch DNA Repair system repairs mismatches in newly replicated DNA strands. Parent strand
T
Correct match
A
Repaired by proofreading
C
Repaired by DNA Mismatch Repair system
G
Mismatch DNA repair
Mismatch DNA repair 3. MutH = an endonuclease • recognizes AMe • nicks the opposite strand. CH3
T~G mismatch in newly synthesized DNA. Which strand must be repaired? Parental GATC = methylation of adenine Repair before the new strand is methylated.
2. MutL attaches& links MutH & MutS
MutL
1. MutS scans DNA & binds to mismatched bases
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Mismatch DNA repair Then: (1) a Helicase (UvrD) unwinds DNA from nick →past the mismatch. (2) an Exonuclease cuts away single bases from nick. (3) SSB - single strand binding proteins. Then DNA polymerase III • fills the site • Ligase seals the gap.
Mismatch DNA repair bacterial
eukaryotic
MutS
hMSH2
MutL
hMLH1, hPMS1, hPMS2
Mutations in these proteins specifically permit random new mutations.
MutH endonuclease recognizes AMe
Helicase Exonuclease SSB Polymerase III Ligase
mutations not known (as yet?)
Significant other functions mutations would be lethal
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