Lecture 6-Mutations, Mutagenesis and DNA Repair PDF

Mutations, mutagenesis and DNA repair Mutant: an organisms or a gene that is different from the wild type Examples: His- yeast or white-eyed Drosophila Lact- in Ecoli unable to metabolize lactose Mutation: any heritable change in the base sequence of DNA Mutagen: a physical or chemical age...

Mutations, mutagenesis and DNA repair Mutant: an organisms or a gene that is different from the wild type Examples: His- yeast or white-eyed Drosophila Lact- in Ecoli unable to metabolize lactose Mutation: any heritable change in the base sequence of DNA Mutagen: a physical or chemical agent that causes mutations to occur  Types of Mutagenesis Point mutation Multiple mutation (single changed base pair) 2-3 changed base pair) Base deletion Base substitution Base addition  Mutagenesis Mutagenesis is the process of producing a mutant Mutagenesis Spontaneous mutagenesis Induced mutagenesis (occurs in nature) (caused by a mutagen) - Natural radiation - Oxygen radicals: chemically modifying DNA (e.g. oxygen radicals convert guanine into 8-hydroxyguanine) -The bulk occurs during DNA replication as a result of errors in base pairing  Types of Mutagenesis Silent (neutral) mutation Missense (nonsense) May cause little of no mutation change in the amino acid Lead to the production of a sequence or in the ability nonfunctional and/or truncated of protein to fucntion polypeptide  Conditional mutations (exhibit the mutant phenotype under certain conditions) Temperature sensitive (ts Termination or nonsense mutant) mutation Protein in active at one If mutation generate a stop temperature but inactive codon at the other - Phenotype is capitalized (Lac+ or Lac-) while the genotype is written in lower case (lac+ or lac-) - (+) means able and (-) means unable for example His+ can synthesize histidine while His- cannot - Amp-r means ampicilin resistant and Amp-s means ampicilin sensitive (the corresponding genotypes are amp-r and amp-s)  Biochemical basis of mutants -Destruction of the three dimentional structure For example: 1) in a protein whose structure is determined by an interaction between one positively charged amino acid (lysine) and negativey charged amino acid (glutamic acid), a substitution of methionine (uncharged) for lysine would destroy the 3-D structure 2) A protein stabilized by hydrophobic structure (substitution of glutamine (polar) for leucine (nonpolar) would be distruptive  Mutation Rates Different types of mutations can occur at different frequencies. For a typical bacterium, mutation rates of 10–7 to 10–11 per base pair are generally seen.  Mutations in RNA genomes Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.  Isolation of mutant: screening versus selection Auxotroph: is mutant that has a nutritional requirement for growth Prototroph: is the parent form from which the auxotroph was derived E-coli with a His- phenotype is said to be histidine auxotrophs Replica plating  Leucine is missing Screening for Leucine auxotrophs lists various kinds of mutants.  1. Base-pair substitutions A point mutation, which results from a change in a single base pair, can lead to a single amino acid change in a polypeptide or to no change at all, depending on the particular codon involved  In a nonsense mutation, the codon becomes a stop codon and an incomplete polypeptide is made. In a missense mutation, the sequence of amino acids in the ensuing polypeptide is changed, resulting in an inactive protein or one with reduced activity.  Destruction of the three dimentional structure 1) in a protein whose structure is determined by an interaction between one positively charged amino acid (lysine) and negativey charged amino acid (glutamic acid), a substitution of methionine (uncharged) for lysine would destroy the 3-D structure 2) A protein stabilized by hydrophobic structure (substitution of glutamine (polar) for leucine (nonpolar) would be distruptive 3) Mutations in the active site destroys the protein Transitions and transversions  2. Frameshifts and other insertions and deletions Deletions and insertions cause more dramatic changes in the DNA, including frameshift mutations, and often result in complete loss of gene function point mutations can be reversible through further mutations but large framshift mutations are not reversible.  Many insertion mutations are due to the insertion of specific identifiable DNA sequences (700-1400bp) called insertion sequences Other types of large scale mutations seems to involve translocations, in which a large section of chromosomal DNA is moved to a new location (of different chromosome) and inversions, in which the orientation of a particular segment of DNA is revered with respect to the surrounding DNA.  3. Transposon and site-directed mutagenesis If insertion of a transposable element occurs within a gene, a loss of the function generally results Because transposable elements can enter the chromosome at various locations, they are widely used as a mutagenic agents  4. Back mutations or reversions Point mutations are typically reversible by the process of reversion A revertant: is a strain in which the wild type phenotype that was lost in the mutant is restored. They are two types 1) same-site revertants:- the mutation that restores activity occurs at the same site at which the original mutation occurred 2) Second site revertants: occurs at a different site Unlike point mutations, large scale deletion mutation are essentially nonrevertable. By contrast, large scale insertions can revert as the result of a subsequent deletion that removes the insertion. But typically, frameshift mutations of any magnitude are difficult to restore to the wild type and mutatnts that carry frameshift mutations are therefore genetically quite stable.  Mutagenesis Mutagenesis is the process of producing a mutant Mutagenesis Spontaneous mutagenesis Induced mutagenesis (occurs in nature) (caused by a mutagen) Chemical mutagens Phyical mutagens A. Chemical mutagens 1. Nucleotide base analogs Nucleotide base analogs  2. Alkylating agents These are chemicals that react with amino, carboxyl and hydroxyl groups subsituting them with alkyl groups Examples Monofunctional Bifunctional (e.g. Ethyl methane sulfonate) (e.g. nitrogen mustards, mitomycin, nitrosoguanidine  2. Alkylating agents They induce base-pair substitution Monofunctional Bifunctional (e.g. Ethyl methane sulfonate) (e.g. nitrogen mustards, mitomycin, nitrosoguanidine Put methyl on G; faulty Cross-links DNA strands; pairing with T faulty region excised by GC AT DNase Alkylating agent differ from the base analogs in that the chemicals are able to introduce changes even in nonreplicating DNA. Base analogs have an effect only when incorporated during DNA replication  3. Intercalative dyes Planar molecules that become inserted between two DNA base pairs and push them apart. During replication, this abnormal conformation can lead to insertions or deletions Examples Acridines Ethidium bromide Induce frameshift mutations 4. Nitrous acid (HNO2) Deaminates A and C 4. Hydroxylamine (NH2OH) Reacts with C Summary of chemical mutagens B. Physical mutagens 1. UV radiation: non-ionizing radiation The shorter the wave length, the higher the energy DNA absorbs light strongly at 260 nm Pyrimidine primers:- A state in which two adjacent pyrimidines (Cytosine or thymine) on the same strand become covalently bonded. During replication the probability of DNA polymerase misreading the sequence at this point is greatly increased 2. Ionizing radiation  2. Ionizing radiation - More powerful form of energy compared to UV and includes short wavelength rays such as (X-rays, cosmic rays and gamma rays) -They cause water (chemical free radcals OH) and other substances to ionize and mutagenic effects are brought about indirectly through this ionization -Free radicals react with and inactivate macromolecules in the cell, of which DNA is most important - at higher doses multiple hits in the DNA occurs leading to the death of the cell - Unlike UV, ionizing radiation penetrates through glass and other materials  Reversion The process in which the wild type phenotype is regained is called back mutation, reverse mutation or reversion  Reversion Spontaneous Induced Same site reversion Different site reversion (true reversion) (Pseudoreversion) Second-site or suppressor mutation Intragenic revertants Revertants of frameshift mutations  Intergenic suppression Refers to a mutational change that eliminates or suppresses the mutant phenotype 1) Mutation in the binding site of a protein a mutation in the binding site of protein A that prevents the protein from interacting with protein B. A second mutation in the binding site of protein B, makes the mutant protein B to bind the mutant protein A Protein A Protein B Mutant protein B Mutant protein A  1) Nonsense mutations mutations in many codons may give rise to the stop codon UAG A phage may have acquired a UAG codon. When phage infects host bacteria, the mutant phage may grow normally. Such bacteria suppress mutation and contain suppressor:- 1) via many mutations in the bacteria or the phage itself 2) The bacteria contains altered tRNA that might contain anticodon CUA, which pairs with the stop codon (Suppressor or nonsense suppressor tRNA)  Ames Test (Reversion as a means of detecting mutagens and carcinogens) -There is a good evidence that human cancers have environmental causes - Chemicals used in industry or agriculture may be carcinogenic - mutagenic is not always carcinogenic - Finding out that a certain chemical is mutagenic in abacterial system serves as a warning of possible danger Mutagenesis test for carcinogens is called Ames test (After Bruce Ames)  Protocol of Ames Test 1) Histidine-requiring His- mutants of Salmonella typhimurium are used to test for reversion to His+ 2) Solid medium containing a very small amount of histidine is prepared 3) A small amount of rat liver extract and anout 108 His- mutant cells are spread on the plates 4) The substance to be tested is added to only one set while distilled H2O is added to the control (no carcinogens) Mutants used for Ames test Histidine-requiring Salmonella enterica AND Tryptophan-requiring Escherichia coli Spontaneous reversion Mutants used for Ames test Histidine-requiring Salmonella enterica Tryptophan-requiring Escherichia coli  Modifications of Ames test - Addition of liver enzyme preparations to convert the chemicals to be tested to their active mutagenic forms. It has been established that many carcinogens are not directly carcinogens themselves but undergo modifications in the human body that convert them into active substances. These changes take place in the liver, where enzymes called mixed-function oxygenases normally involved in detoxification cause the formation of epoxides or other activated forms of the compounds  DNA repair mechanisms 1) Proofreading activity of the polymerase enzymes 2) Mismatch repair 2. Mismatch repair 2. Mismatch repair  3. DNA repair of depurination Repaired by enzmes called AP endonucleases (AP=apurinic)  4. DNA repair of deamination A cytosine losing an amino group becomes uracil Phosphodiesteras removes the suager phosphate residue Another glycosylase Acts on apurinic removes hypoxanthine or baseless (Adenine deamination) sites)  4. DNA repair by direct reversal Pyrimidine dimers Repaired by the enzyme photolyase, which is activated by visible light (300-600 nm) and cleaves the dimers to yield intact pyrimidines  5. DNA repair by excision repair A mechanism by which pyrimidine dimers can be repaired without photolyase or in case of bulky insertions A repair endonuclease recognizes the distortion and makes two cuts (12- 13 bp apart, 8 to 5‘ side and 4-5 to the 3‘ side)  5. DNA repair by excision repair 1) In E-coli three genes are invloved to produce the incision (uvrA, uvrB, and uvrC) but in mammalian, many more genes 2) Xeroderma pigmentosum (XP) is a syndrome in which patients are sensitive to sunlight and have the tendency to develop skin cancer Cause:- mutation in any one of seven different genes invloved in various steps in the human excision-repair process 3) Cockyne‘s syndrome: also sensitivity to UV light including neurological and developmental abnormalities cause:- genes invloved in the excision-repair pathway  6. Recombinational repair Effect of thymine dimer on DNA replication:- 1) When polymerase III reaches a thymine dimer, the replication fork fails to advance 2) A thymine dimer is still capable of forming hydrogen bonds with two adenines 3) But the dimer introduces a distortion into the helix 4) When an adenine is added, polymerase III reacts to the distorted region as if a mispaired base had been added 5) The editing fucnction then removes the adenine 6) The cycle begins again and the result is that the polymerase is stalled at the site of the dimer Sister strand exchange  7. SOS response Some DNA damage can lead to cell death if not repaired. A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage and initiates a number of DNA repair processes, both error-prone and high-fidelity.  7. SOS response some mutations can be inherited but some can not (e.g. pyrimidine dimers) If pyrimidine dimers can not be repaired, cell can not replicate and they die DNA repair can be error-free but can also be error-prone (SOS response) SOS is activated as a response to some types of DNA damage  7. SOS response Lex A: gene repressing the SOS system Rec A: gene inactivates Lex A, it is a protease that is activated as a result of DNA damage The interaction of LexA and Rec A, gover the expression of - uvr A and uvr B invloved in excision repair - umuC and umuD required to help replication bypass the offending lesion