Presentation on theme: "DNA Mutation and Repair"— Presentation transcript:
1 DNA Mutation and Repair Lecture 5 ofIntroduction to Molecular Biology生理所 蔡少正
2 DNA DamageMechanisms for maintaining genetic stability associated with DNA replication in E. ColiMachanism Cumulative error frequencyBase pairing ~DNA polymerase actions (including base ~selection, 3'->5' proofreading)Accessory proteins (e.g. SSBP) ~10-7Post-replication mismatch correction ~10-10
3 Spontaneous alterations: (a) Mismatches: Occurs during DNA synthesis (i.e. replication, repair, or recombination)
4 (b) Tautomeric shiftsNucleotides spontaneously under go a transient rearrangement of bonding, e.g. a shift from NH2 (amino form) to NH (imino form) or C=O (keto) to C-OH (enol). Therefore, if any base in a template strand exists in its rare tautomeric form during DNA replication, misincorporation in the daughter strand can result.
6 (c) DeaminationThree of the four bases normally present in DNA (cytosine, adenine, and guanine) contain amino group (NH2). The loss of the amino group (deamination) can occur spontaneously and result in the conversion of the affected bases to uracil, hypoxanthine, and xanthine, respectively.
7 (d) Loss of basesDepurination and depyrimidination: The loss of purines or pyrimidines from DNA usually occurs at acidic pH; however, it can also happen in physiological pH (~10,000 purine per day in mammalian cell; ~500 pyrimidine/day). This will results in breaking the 3' phosphodiester bond called b-elimination.
8 Induced Mutations (a) Physical agents that damage DNA: --- Ionizing radiation: OH, O2-, H2O2, damage base and sugar residues.--- UV radiation: Cyclobutane pyrimidine dimers, Thymidine dimers (T-T) dimer
9 Chemical Agents G-C ---> G*T --->A-T T-A --->T*-G ---> CG (b) Chemical agents that damage DNA:--- Alkylating agents: Alkylating agents are electrophilic compounds with affinity for nucleophilic centers in organic macromolecules. These include a wide variety of chemicals, many of which are proven or suspected carcinogens (such as nitrous acid, hydroxylamine, and ethylmethane sulfonate, EMS), Adding alkyl group to hydrogen-bonding oxygen of G or T, resulting in G-T mispairingG-C ---> G*T --->A-TT-A --->T*-G ---> CG
10 Base-analogue AgentsA base analogue is a substance other than a standard nucleic acid base that can be incorporated into a DNA molecule by the normal process of polymerization. Such a substance must be able to pair with the base on the complementary strand being copies, or the 3'->5' editing function will remove it. For example, 5-bromouracil is an analogue of thymine and might cause an A-T to G-C transition mutation.
12 Intercalating Agents: Intercalating agents: Substances whose dimensions are roughly the same as those of a purine-pyrimidine pair. In aqueous solutions, these substances form stacked arrays, and are also able to stack with a base-pair by insertion between two base-pairs. This may result in frameshift mutation.
13 Model of intercalating agent induced mutagenesis
14 Metabolite MutagensChemicals that are metabolized to electrophilic reagents: Aflatoxins, benzo[a]pyreneA mutagen is a physical or chemical agent that causes mutations to occurs.Mutagenesis is the process of producing a mutation.Mutant refers to an organism or a gene that is different from the normal or wild type.
15 Reversion and the Ames test: Mutants may have second mutation and become wild type again.Reversion was used as a means of detecting mutagens and carcinogens- the Ames test
16 DNA Repair Mechanisms(1) Repair by direct reversal: The simplest mechanism. e.g. UV induced T-T dimer is recognized by photolyase and is cleaved into intact thymine (light dependent). This is called photoactivation
17 Excision Repair(2) Excision Repair: The most ubiquitous repair mechanism, which can deal with a large variety of structural defects in DNA.
18 Recombinational Repair (3) Recombinational repair (Postreplicational repair): Occurs before excision repair has happened or when excision repair can not fix the problem
19 The SOS response(4) The SOS response: The SOS response system is only active in response to some signal such as a blocked of replication fork. In E. Coli, recA and lexA govern the expression of a number of other genes involved in DNA repair. This is an error-prone DNA repair mechanism and result in higher than normal mutagenesis.
20 SOS DNA Repair 1. DNA damage 2. RecA converted to RecA* 3. RecA* facilitated LexA self-cleavage4. Increased synthesis of SOS proteins5. Error prone repair induced6. DNA damage repaired7. RecA* returned to RecA8. LexA no longer self-cleaved9. LexA repressed SOS genes10. LexA repress lexA gene expression
21 Type of Mutations(I) I. Point mutation: A. Base substitution Change in DNATransition: One purine replaced by a different purine;or one pyrimidine replaced by a diferent pyrimidineA G T CTransversion: A purine replaced by a pyrimidine or vice versaA TC G
22 Type of Mutations (II)Change in protein1. Silent mutation: altered codon codes for the same a.a.2. Neutral mutation: altered codon codes for functional similar a.a.3. Missense mutation: altered codon codes for different dissimilar a.a.4. Nonsense mutation: altered codon becsomes a stop codonGAG (Glu) --->GAA (Glu)GAG--->GAC (Asp)GAG ---> AAG (Lys)GAG ---> UAG (stop)
23 Type of Mutations (III) B. Frameshift mutation: addition or deletion of one base-pair result in a shift of reading frame and alter amino acid sequence1. Wild type: ATG ACC AGG TC2. Base addition: ATG ACA CAG GTC3. Base deletion: ATG ACA GGT CArgMetThrMetThrGlnValMetThrGly
24 Type of Mutations (IV) II. Insertion III. Deletion IV. Translocation V. Inversion