Mutation and Repair BBT317 PDF

Mutation  Mutation A stable and heritable change in a DNA sequence Create a phenotype different from that normally expressed by the wildtype allele of that gene. – Base substitutions, – Duplications, – Deletions – Chromosomal aberrations (invarsions,...

Mutation  Mutation A stable and heritable change in a DNA sequence Create a phenotype different from that normally expressed by the wildtype allele of that gene. – Base substitutions, – Duplications, – Deletions – Chromosomal aberrations (invarsions, translocations) Classification of Mutations in Genetics Dominant and Recessive Wildtype = +, Mutation =m1(recessive) or M2 (dominant) 1. +/+ gives wildtype phenotype 2. +/m1 gives wildtype phenotype 3. M2/+ gives Mutant phenotype Semidominant a dominant mutation displays a more severe (or extreme) phenotype as a homozygote than heterozygote. – M3/M3> M3/+>+/+  Muller's Classification of Mutants H. J. Muller, 1932 Based on comparing the phenotypic effects of a mutation in homo-, hetero-, and hemizygotes. 1. Nullomorphs, 2. Hypomorphs, 3. Hypermorphs, 4. Antimorphs, and 5. Neomorphs  Nullomorphs Mutants with no remaining gene function→no functional gene product, equivalent of a full deletion of the gene in terms of its influence on the final phenotype Null allele Defects in gene expression – Transcriptional nulls – Translational nulls – Inactivating nulls Most nullomorphic mutants are recessive, i.e. enzymes Mutations in genes that code for structural or highly concentration dependant gene->dominant  Hypomorphs Hypomorphs are mutants that produce some degree of residual activity, but not enough to provide wildtype activity in mlm homozygotes. Weak allele Most hypomorphic mutants are recessive Give enough product to allow survival, but not enough to produce a normal phenotype.  Hypermorphs Hypermorphs produce either a hyperactive protein product or a harmful excess of the normal protein product mlm > m/+ > m/Df. (hypermorph) Hypermorph can be valuable tool for dissecting a genetic process Hypermorph can be valuable tool to identify specific functions of a group of functionally redundant genes  Antimorphs An antimorphic mutation results in a protein product that antagonizes, or poisons, the wildtype protein Anti·morphic mutations are dominant Increasing the dose of the wildtype allele can often ameliorate the phenotype of an antimorphic mutant. One should be able to revert ( or more precisely ~pseudo-revert") an antimorphic mutant to a nullomorphic mutation of the same gene A/A > A/Df> A/+ >>> +/Df > +/+ (Antimorph) mlm > m/+ > m/Df. (hypermorph)  Neomorphs A mutation that causes a gene to be active in an abnormal time or place. Burkitt's lymphoma A dominant mutation (Antp73b) in Drosophila that causes the antennae to be replaced by legs  Modern mutant terminology Mutants that reduce the level of gene product are often classified only as loss-of-function mutants – Null mutants – Partial loss-of-function mutants (amount or functon) – Temperature-sensitive (or other conditional) loss-of-function mutants  Modern mutant terminology Classification of Dominant Mutants – Dominant Negative (Poisonous product) – Gain-of-Function mutant (Inappropriately regulated) – Heterochronic Mutant (Wrong time)  DNA-level terminology 1. Base pair substitution mutants 2. Missense mutants 3. Nonsense mutants 4. Silent substitutions 5. Base pair inserions or deletions 6. Frameshift mutants Base pair substitution mutants  The Importance of Mutations Mutation is the source of all genetic variation – raw material of evolution Adaptation to the changing environment Mutations serve as important tools of genetic analysis  Why looking for new mutants??? ✓ To identify genes required for a specific biological process ✓ To isolate more mutations in a specific gene of interest ✓ To obtain mutation tools for structure-function analysis ✓ To isolate mutations in a gene so far identified only by molecular approaches  Mutations in Multicellular organisms Two broad categories 1) Somatic 2) Germ-line ✓ Somatic mutations: Time, Rate, Effect ✓ In single-cell organisms, however, there is no distinction between germ- line and somatic mutations, because cell division results in new individuals ✓ Gene and chromosome mutation. Types of Gene Mutations The number of possible transversions is twice the number of possible transitions, but transitions usually arise more frequently.  Phenotypic effects of mutations Loss-of-function mutations Gain-of-function mutation Terms to know – Wild-type phenotype – Forward mutation, & Reverse mutation – Neutral mutation  Suppressor mutations Intragenic Compensatory changes in the protein Intergenic Suppressor mutations  Mutation rate The frequency with which a wild-type allele at a locus changes into a mutant allele is referred to as the mutation rate. The mutation rate provides information about how often a mutation arises. Generally expressed as the number of mutations per biological unit, which may be mutations per cell division, per gamete, or per round of replication. Mutation frequency is defined as the incidence of a specific type of mutation within a group of individual organisms.  Mutation Rates Three factors affects mutation rates – The frequency with which primary changes take place in DNA – The probability – Our ability to calculate mutation rates Viral and Bacterial:1 to 100 mutations per 10 billion cells (1X10-8 to 1X10-10) Eukaryotic genes 1 to 10 mutations per million gametes (1X10-5 to 1X10-6) – The mutation rate for achondroplasia (a type of hereditary dwarfism) is about 4 mutations per 100,000 gametes, usually expressed more simply as 4X105 – For achondroplasia, the mutation frequency in the United States is about 2X104, which means that about 1 of every 20,000 persons in the U.S. population carries this mutation The higher values in eukaryotes may be due to the fact that the rates are calculated per gamete, and several cell divisions are required to produce a gamete, whereas mutation rates in prokaryotic cells and viruses are calculated per cell division.  Causes of Mutations Spontaneous Replication Errors – Tautomeric shifts – Wobble formation – Strand slippage – Unequal crossing over Spontaneous Chemical Changes – Depurination – Deamination Tautomeric shifts Mispairing Wobble formation→ Incorporated error The original incorporated error leads to a replication error, which creates a permanent mutation, because all the base pairings are correct and there is no mechanism for repair systems to detect the error. Strand slippage Unequal crossing over  Expanding trinucleotide repeats Mutations in which copies of a trinucleotide may increase greatly in number are called expanding trinucleotide repeats. Strand slippage in DNA replication and crossing over between misaligned repeats are two possible sources of expansion.  Depurination Depurination is a common cause of spontaneous mutation; a mammalian Deamination cell in culture loses approximately 10,000 purines every day.  Chemically Induced Mutations Mutagen Chemicals can produce mutations by a number of mechanisms. – Chemical Base analogs are inserted into DNA and Base analogs frequently pair with the wrong base. Hydroxylamine Alkylating agents, deaminating chemicals, Alkylating agents hydroxylamine, and oxidative radicals change the structure of DNA bases, Deamination thereby altering their pairing properties. Oxidative reactions Intercalating agents wedge between the Intercalating agents bases and cause single-base insertions and deletions in replication. Radiation Base analogs Ethyl group to T and G Deaminates C to U & A to HX Only Cytosine EXTRA SLIDE EXTRA SLIDE Oxidative reactions How natural processes can change the information stored in DNA Radiation How natural processes can change the information stored in DNA  What Causes Mutation ❑ Spontaneous mutation ❑ If mutations occurred spontaneously, then the mutations might be expected to occur at different times in different cultures; so the resulting numbers of resistant colonies per culture should show high variation (or “fluctuation”) ❑ The early mutations gave the higher numbers of resistant cells because they had time to produce many resistant descendants. The later mutations produced fewer resistant cells  Luria and Delbrück fluctuation test Mutations Are Chance Occurrences Modifying the Genome at Random  Detection of Spontaneous Mutations Luria and Delbrück fluctuation test and replica platting Detection of Spontaneous Mutations  Ames Test TESTING FOR MUTAGENICITY 1974, Bruce Ames Both cancer and mutations result from damage to DNA Salmonella typhimurium – Defects in the lipopolysaccharide coat – DNA repair system has been inactivated One of the 4 strains used in the Ames test detect base-pair substitutions; the other three detect different types of frameshift mutations Only bacteria that have undergone a reverse mutation of the histidine gene (his-:his+) are able to synthesize histidine and grow on the medium.  DNA Repair ❑ Cause of DNA Damage????? ❑Radiation, chemical mutagens, heat, enzymatic errors, and spontaneous decay ❑ Rate of DNA damage Vs rate of DNA Repair ❑fewer than one in a thousand DNA lesions becomes a mutation; all the others are corrected ❑ Lower rate of Mutation ❑is indicative of the efficiency of these repair systems ❑ The repair Pathway ❑chemically repairs the damage to the DNA base ❑is redundant (Many types of DNA damage can be corrected by more than one pathway of repair) ❑deletes the damaged DNA and uses an existing complementary sequence as a template to restore the normal sequence  DNA Repair ❑Direct reversal of damaged DNA ❑Homology-dependent repair systems ❑Excision-repair Pathways ❑Base excision Repair ❑Nucleotide excision repair ❑Mismatch-repair System (Post-replication) ❑Repair of double-strand breaks ❑NONHOMOLOGOUS END-JOINING ❑HOMOLOGOUS RECOMBINATION  Direct reversal of damaged DNA ENZYMATIC REPAIR Alkyltransferases ❑The methyltransferase from E. Coli enzyme transfers the methyl group from O-6-methylguanine to a cysteine residue on the protein. PHOTOREPAIR ❑The transfer inactivates the enzyme, so this repair system can be saturated if the level of alkylation is high enough.  Base excision repair ❑Numerous DNA glycosylases exist. Each removes a specific type of modified base by cleaving the bond that links that base to the 1- carbon atom of deoxyribose. ❑Uracil glycosylase recognizes and removes uracil produced by the deamination of cytosine ❑AP (apurinic or apyrimidinic) endonuclease ❑Hypoxanthine, 3-methyladenine, 7-methylguanine Homology-Dependent Repair Systems Base excision repair  Base excision repair ❑ Bacteria use DNA polymerase I to replace excised nucleotides ❑ Eukaryotes generally use DNA polymerase β, which has no proofreading ability and tends to make mistakes ❑ DNA polymerase β makes one mistake per 4000 nucleotides inserted. ❑ About 20,000 to 40,000 base modifications per day are repaired by base excision ❑ DNA polymerase β may introduce as many as 10 mutations per day into the human genome ❑ How are these errors corrected?  Homology-Dependent Repair Systems Nucleotide excision repair Distortions in the DNA backbone Pyrimidine dimers or large hydrocarbons attached to the DNA, bulky DNA lesions which distorts the DNA Found in cells of all organisms from bacteria to humans and is one of the most important of all repair mechanisms In yeast, approximately twelve genes of RAD3 groups are involved In human beings, twenty-five proteins are involved; they remove twenty-seven to twenty-nine nucleotides, as compared to twelve to thirteen in E. coli  Homology-Dependent Repair Systems (20 proteins) Nucleotide excision repair ❑DNA repair systems in eukaryotes are highly conserved from yeast to humans ❑Yeast repairsome recognize damaged DNA ❑How can the repairosome “know” which strand of a gene is transcribed and which is not? ❑ Repair in non dividing cells. ❑Transcription factor TFIIH is involved in repair of UV damage; it has helicase ❑activity and is found in both processes Transcription-coupled repair in eukaryotes  POSTREPLICATION REPAIR ❑ Replication errors failed to be corrected by the 3-to-5 proofreading function of the replicative polymerase ❑ Mismatch-repair system, can detect such mismatches, 1. Recognize mismatched base pairs 2. Determine which base in the mismatch is the incorrect one 3. Excise the incorrect base and carry out repair synthesis ❑ DNA methylation in Bacteria ❑ Adenine Methylase, methylates position 6 which does not interefere with base pairing activity. ❑ Delay in the methylation  Mismatch-repair System Mismatch repair in E. Coli ❑Excision repair triggered by mismatches is referred to as mismatch repair, which encompasses about 99% of all DNA repairs ❑The mismatch repair system, follows behind the replicating fork ❑The genes are called mut ❑The mutU gene is also known as uvrD. Mismatch-repair System Mismatch repair in human An important target of the human mismatch repair system is short repeat sequences that can be expanded or deleted during replication by the slipped mispairing mechanism  Repair of double-strand breaks ❑Double-strand breaks can arise Nonhomologous End-joining spontaneously (for example, in response to reactive oxygen species), or they can be induced by ionizing radiation. ❑Two distinct mechanisms are used to repair these potentially lethal lesions: Nonhomologous end joining and Homologous recombination ❑Interestingly, the ability of double-strand breaks to initiate chromosomal instability is an integral feature of some normal cellular processes that require DNA rearrangements. Antibody gene Homologous Recombination  Repair of double-strand breaks Homologous Recombination ❑The mechanism of homologous recombination utilizes the sister chromatid to repair double-strand breaks. ❑A double-strand break induces an enzyme to chew back 5` ends, leaving 3` overhangs that are coated with proteins, including RAD51, a RecA homolog ❑The coating of these regions with proteins that include the RecA homolog, RAD51 ❑A segment of the sister chromatid (blue) is used as a template to repair the break ❑The RAD51–DNA filament then takes part in a remarkable search of the undamaged sister chromatid for the complementary sequence that will be used as a template for DNA synthesis Nature Reviews: Genetics 2, 2001, 196–206  Faulty DNA Repair ❑ Xeroderma pigmentosum ❑ autosomal recessive trait ❑ an inability to repair thymine dimerization induced by UV light ❑ a high incidence of skin cancer ❑ seven genes are required for nucleotide- excision repair in humans ❑ Li-Fraumeni ❑ p53 (halt cell division & stimulate DNA repair) ❑ Initiate apoptosis ❑ Predispositon to many different types of Cancer Faulty DNA Repair