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Mutagen
Spontaneous
Mechanism
DNA replication; repair errors, spontaneous modification of nucleotides Pyrimidine dimers induce error prone repair (SOS) Base analog Base analog Alkylating agent, generates O6methyl guanine Alkylating agent, generates N4hydroxycytosine DNA intercalating agent Ionization of molecules into free radicals and reactive ions
Type of mutation
All types of mutations
UV irradiation 2-aminopurine (2-AP) Bromouracil Ethylmethane sulfonate (EMS) & N-methyl-N'-nitroN-nitrosoguanidine (MNNG) Hydroxylamine (NH2OH) Acridine dyes (acridine orange, proflavin) Ionizing radiation (X-rays, rays, cosmic rays)
Mainly G-C to A-T transitions A-T to G-C and G-C to A-T transitions G-C to A-T and A-T to G-C transitions G-C to A-T transitions
G-C to A-T transitions when used in vitro Frameshift probably due to slippage during replication Point mutations; disrupts integrity of chromosomes
Ames Test
To assess the mutagenicity of compounds
Filter paper containing ethyl methanesulfonate (EMS)
Classes of Mutations
Point mutations
a) Transitions, in which a purine (or pyrimidine) is replaced by another b) Transversions, in which a purine is replaced by a pyrimidine or vice versa
2.
Insertion/deletion mutations
Insertion mutation
Met Thr Asp Glu - ATG ACC GAC GAG - TAC TGG CTG CTC - Met Thr Asp Arg Arg Glu - ATG ACC GAC CGA CGA GAG - TAC TGG CTG GCT GCT CTC - -
Deletion mutation
Met Thr Asp Glu - ATG ACC GAC GAG - TAC TGG CTG CTC - Met Asp Glu - ATG GAC GAG - TAC CTG CTC - 7
Nitrous acid (HNO2) oxidatively deaminate primary amines Hence, A=T GC and GC A=T transitions Nitrite (the conjugate of HNO2) is used as a preservative in prepared meats
Alkylating agents such as dimethyl sulfate, nitrogen mustard, ethylnitrosourea, and MNNG generates transitions
DNA exposed to MNNG yields O6-methylguanine residues which can base pair with either C or T.
The distance between 2 consecutive base pairs is doubled by the intercalation of such molecules Replication of such DNA results in deletion or insertion of one or more nucleotides in the newly synthesized DNA Frameshift mutation
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Met Thr Arg Arg - ATG ACC CGA CGA G - TAC TGG GCT TCT C - -
Met Thr Glu Stop - ATG ACC GAC TAG - - ATG AAA TAC TGG CTG ATC - - TAC TTT
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If left uncorrected, such changes could lead to deletion or substitution of base pairs during DNA replication
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Deamination of bases in DNA yields an unnatural base which can be directly recognized and removed by a specific DNA glycosylase E.g. deamination of C produces U, which can be repaired by uracil DNA glycosylase
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About 3% of C nucleotides in vertebrates are methylated to help in controlling gene expression Accidental deamination of 5-methyl-cytosines yields thymines, thus resulting in mismatched base pairing
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Formation of a dimer between 2 pyrimidine bases when cells are exposed to UV irradiation Occurs between two adjacent thymine or cytosine bases Repaired by nucleotide excision repair system
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DNA Damage
Cancer
DNA Repair
Aging
DNA repair .
A major defense against environmental damage to cells Minimizes cell killing, mutations, replication errors, persistence of DNA damage and genomic instability Abnormalities in these processes have been implicated in cancer and aging
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Spontaneous damage
Depurination Deamination
Complementary base pairing alone is not sufficient to determine fidelity Rare tautomeric forms of the 4 bases (A, T, G, C) occur transiently (1:104 or 1:105). Rare tautomeric form of C can also pair with A
Proofreading by DNA polymerase decreases errors introduced during DNA synthesis by 1000-fold Strand-directed mismatch repair reduces the errors by a further 100-fold
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DNA Pol has a higher affinity for correct nucleotide because of base pairing requirement 35 proofreading exonuclease activity of DNA Pol removes mismatched nucleotide at 3OH end of primer strand
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During DNA replication, mismatch repair corrects errors that remain after proof-reading In eukaryotes, newly synthesized DNA are recognized by presence of many nicks Defects in the human mismatch repair system result in a high incidence of cancer e.g. hereditary nonpolyposis colorectal cancer
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In E. coli, mismatch repair involves the MutSLH system Depends on the methylation of selected A residues in GATC to distinguish newly synthesized vs template DNA An endonuclease makes a nick on 5 side of the unmethylated GATC UvrD (helicase) + exonuclease removes defective strand The unmethylated DNA strand is corrected by DNA Pol III
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Removes DNA modification (thymine dimers) Light-dependent reaction Occurs in bacteria but not in eukaryotes Involves a photoreactivation enzyme (PRE) cleaves bonds between thymine dimers
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(D)
Excision Repair
Involves 3 steps
Recognition & removal of distortion or error by a nuclease Gap filling by DNA polymerase Sealing of nick by DNA ligase
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Removes damaged base Many types of DNA glycosylases, each of which cleaves the glycosidic bond leaving a deoxyribose residue with no attached base - apurinic or apyrimidinic sites (AP sites) Most common is uracil-DNA glycosylase which removes uracil (arising from spontaneous deamination of cytosine)
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UvrABC endonuclease
Corrects pyrimidine dimers and other DNA lesions in which the bases are displaced from their normal position or have bulky substituents. In E. coli, NER is an ATP-dependent process involving UvrA, UvrB, UvrC and UvrD proteins In humans, NER requires >16 proteins. Individuals with xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are unable to repair UV-induced DNA lesions XP individuals have 2000-fold incidence of skin cancer
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UvrD helicase
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When a lesion is encountered during replication, Pol III is replaced by error-prone a translesion DNA polymerase, Pol IV (DinB) or Pol V (UmuD2C) Translesion DNA polymerase extends DNA synthesis beyond thymine dimer independent of base pairing and has no proofreading exonuclease activity
Pol III
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(contd)
Pol III then replaces translesion DNA Pol to complete DNA replication Translesion DNA synthesis is error-prone. In E. coli, translesion polymerases are invoked as a last resort as part of the SOS response Individuals with a variant form of XP exhibit are defective in translesion DNA synthesis. They have normal NER proteins but show increased rates of skin cancer
Pol III
Pol V
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In response to DNA damage, RecA triggers the SOS system and induces 43 genes involved in recombinational repair Proteolytic activity of
DNA Pol IV and V are error prone translesion polymerases UvrABCD are involved in NER
Cleavage of LexA repressor induces the expression of many target genes
LexA recA
LexA, RecA and other target genes involved in general homologous recombination and recombinational repair are induced
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emergency repair
Figure 5-51 Molecular Biology of the Cell ( Garland Science 2008)
DSB repair by nonhomologous end joining (NHEJ) is common in mammalian somatic cells Ku is a key protein in NHEJ
Homologous end-joining
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Double strand break is generated when the replication fork encounters a singlestrand nick in the template DNA DSBs are also induced by ionizing radiation, replication errors, oxidizing agents and certain cellular metabolites
DSB is generated
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Reading
Alberts et al. Molecular Biology of the Cell 5th Ed. (2008), Chapter 5, p295-304 Voet & Voet, Fundamentals of Biochemistry 2nd Ed (2006), Wiley Chapter 24
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