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DNA Mutation Mutation is the process by which gene (chromosome) changes structurally In 1943 Luria and Delbruck used the fluctuation test to demonstrate that phenotypic variant in bacteria is due to mutation Enrichment media in a perti dish was plated with E. coli in presence of phage T1 Under normal circumstances there will be no bacterial colonies as all bacteria will be lysed When resistant bacterial grow Genetic fine structure Complementation If two recessive mutations arise independently and if they do so there is there is complementation between the mutants To test complementation test mutant homozygotes are crossed If mutants are allelic (affect same gene) both copies of the gene are mutant, resulting in a mutant phenotype If nonallelic there is a wide type phenotype Mutation that fails to complement each other are called functional alleles Figure 1. Complementation test to define allelism. (a) Shows allelic mutants affecting same gene and (b) mutants are nonallelic Mutation Mutationrates rates Mutation rate is the number of mutations that arise per division in bacteria and Mutation rate isorganisms, the numberorofthemutations that arise perthat division single-celled number of mutations arise in perbacteria gameteand in single-celled organisms, or the number of mutations that arise per gamete in higher organisms. higher organisms. Point mutations These consist of single changes in the nucleotide sequence and they could be replacement, addition or deletion A second point mutation in the same gene can result in double mutation, reversal to original or intragenic suppression (addition of a base followed by deletion of a nearby base) Mutations will result in new amino acids and new proteins appear that can alter the morphology or physiology of the organism and result in phenotypically novel or lethal mutants Figure 2. Types of point mutations on DNA showing singlestep (replacement, addition deletion) and double step (double mutation, reversal to original or intragenic suppression Frameshift mutations Point mutations that add or subtract a base are potentially the most devastating because they change the reading from the site of mutation onward and they are called frameshift mutations Frameshift causes two problems 1) All codons from the frameshift on will be different and most likely yield a useless protein 2) Stop signal are misread and so there may either be premature stoppage of translation or translation may proceed beyond the gene Figure 3. A frameshift mutation showing possible effects of insertion of a single base resulting in the creation of a new stop sequence on the mRNA Back Mutation and suppression A second point mutation on the same gene can have three effects – Mutation can result in another mutant codon or one codon that has experienced two changes (double mutation) – If mutation is at the same site, the original sequence can be returned, an effect known as back mutation – Intragenic suppression can occur and this occurs when a second mutation in the same gene masks the occurrence of the original mutation without actually restoring the original sequence. The new sequence is a double mutation but with the same phenotype Suppressed mutations can be distinguished from back mutations through genetic crosses and DNA sequencing Conditional lethality These are mutants that are lethal under certain conditions but not under others Nutritional-requirement mutants reflect failure of one or more enzymes in the biosynthetic pathways of the bacteria Temperature-sensitive mutants are another class they are normal at 25 oC (permissive temperature) but cannot make DNA at 42 oC (restrictive temperature) Spontaneous mutagenesis If the base of a DNA underwent a proton shift into one of its rare tautomeric forms (tautomeric shift) during replication, an inappropriate pairing of bases would occur If a purine (or pyrimidine) is replaced by another purine (or pyrimidine) through a translational state involving a tautomeric shift this is referred to as transition mutation The form of replacement in which purine replaces a pyrimidine or vice versa is referred to as transversion mutation. Chemical mutagenesis Mutation can be caused by (a) Radiation (b) Chemicals (3) Temperature (4) Enzymatic errors and (5) Spontaneous decay Mice or rats are used to determine if cexpensive and time consuming Ames test was introduced to find out whether new chemicals are mutagenic. S. typhimurium requires histidine and so will not growth in a minimal medium. However, the strain will grow in minimal medium if a mutagen is present because it causes histidine pathway to revert to wild type. So if an agent is mutagenic S. typhimurium will grow in minimal medium Chemical mutagenesis Translations Translations are routinely produced by base analogues. Two examples of these analogues are pyrimidine analogue 5-bromouracil (5BU) and purine analogue 2aminopurine (2AP). Both act the same and target the base thymine. They also targets guanine Transversions Ethyl methane sulfonate and ethyl ethane sulfonate causes removal of the purine ring and DNA polymerase III will be free to insert any of the four bases. If thymine is placed the original base pair is restored, insertion of cytosine will result in transition while insertion of adenine or guanine will result in transversion Insertions and deletions These are caused by molecules of acridine dyes, such as proflavin and acridine orange Misalignment mutagenesis Additions or deletions in DNA can occur by misalignment of a template strand and the newly (progeny) formed strand in a region in which there is a repeated sequence. Intergenic suppression When mutation occurs, back mutation or intragenic suppression will lead to the survival of the individual A third route is intergenic suppression which is the restoration of the function of a mutated gene by changes in a different gene called, suppressor gene Suppressor genes are usually tRNA and when mutated they change the way in which a codon is read DNA repair Thousands of bases of DNA are damaged each day. Cells have evolved mechanisms to repair and these mechanisms are placed in three broad categories (1) Damage reversal (2) Excision repair (3) Postreplicative repair Damage reversal In E. coli DNA photolyase normally binds in the dark to create thymine-thymine dimmers. When light shines this enzyme will break the dimmer bonds with light energy thus reversing dimerization Excision repair This is the repair in which damaged bases are removed and patched. There are three types of this repair (a) UV damage repair (b) AP repair (c) mismatch repair Excision repair UV damage repair When DNA is exposed to UV dimmers are formed. An endonuclease enzyme known as ABC exinuclease will hydrolyze the damaged strand DNA helicase II will separate the strands and DNA polymerase I and ligase will fill in the gap Excision repair AP repair This is the repair of the apurinic and a pyrimidinic sites on DNA. These are sites in which a base has been removed by radiation or DNA glycosylases (enzyme that sense damaged DNA and remove it). AP endonucleases will nick the DNA. An exonuclease will remove the short length of DNA and DNA polymerase I and ligase will repair the patch Excision repair Mismatch repair This is responsible for 99% of all repairs to DNA. As DNA polymerase replicates DNA some errors occur that are not corrected by the proof reading .These errors are corrected by the mismatch repair system whose members are specified by mutatorH, mutatorL, mutatorS and mutatorU genes This system works such that the mismatch is recognized by the products of the mutatorL and mutatorS genes. The product of mutatorH nicks the DNA. While the product of mutatorU gene (also called uvrD), DNA helicase II unwinds the nicked region so it falls free. DNA polymerase I and ligase will then patch up Postreplicative repair When E. coli DNA polymerase encounters some damages, such as thymine dimmers, it stops, skips down the DNA, leaving a gap before starting replication Repairing of this gap is done by a group of enzymes at the recA locus Since repair occurs due to failure of replication the process is called postreplication failure it is also known as recombinational repair Recombination This is a breakage and reunion process. Homologous parts of chromosome come into apposition, at which point both strands are broken and then reconnected in a crosswise fashion Figure 4. Crossover of homologous chromosomes during meiosis between the loci Holliday mechanism of breakage and repair reunion Two homologous DNA will form a duplex Breakage of the duplex, each at the same place and only on one strand of each duplex The broken strands then pair with the complement on the other duplex and covalent bonds are formed Branch migration occurs In order to release cross-linked duplexes, a second cut and it occurs in the previously uncut strand Figure 5. The Holiday model of genetic recombination