PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Figure 15-20Figure 15-21Slide 22Slide 23Slide 24Figure 15-5Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Lecture 24October 24, 2014Mutations and DNA repairMutationsTypes of mutations (at the DNA level)Single-gene mutation Chromosome mutations Mutations in chromosome numberCells affected by mutationGermline cells - Mutation passed on to offspringSomatic cells - only inherited to daughter cells (clone)e.g. may lead to cancerCauses of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous' Effects of mutations -> Protein -> Gene expression1. Spontaneous DNA damageChemical (deamination, depurination)Errors in DNA replication - Tautomeric forms of bases- Replication slippage2. Induced DNA damage (mutagens) Physical: Radiation: UV, X-rays (e.g. Computed tomography (CT) scans)Chemical: Alkylating agents, nitroso-compounds, reactive oxygenBiological: Transposons, T-DNACauses of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous'Causes of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous' Ionizing radiation DNA damageSources of ionizing radiation impacting humansMutagen classes: Base analogs, alkylating agents, X-rays, UV, intercalating agents-> Spectrum of mutations variesMeasurement of mutation rates1. Mutation from ad+ to ad- (adenine prototroph to adenine auxotroph)2. Survival********Normal basepairing1. Spontaneous mutation by deamination!Spontaneous mutations - II Abnormal basepairing by tautomeric forms of the bases C and T-> P~0.0001 -> Mutations during replication. -- Repair?2. Mutations induced by mutagens: Chemical Example: Alkylating agent ethylmethanesulfonateCCCAAMutations induced by radiation: UV light causes pyrimidine dimers (T-T)Result: Errors in DNA replicationChemical Mutagens - Intercalating agents ...... stretch the DNA … causing insertionsduring DNA replication-> frameshift mutations1. Spontaneous DNA damageChemical (deamination, depurination)Errors in DNA replication - Tautomeric forms of bases- Replication slippage2. Induced DNA damage (mutagens) Physical: Radiation: UV, X-rays (e.g. Computed tomography (CT) scans)Chemical: Alkylating agents, nitroso-compounds, reactive oxygenBiological: Transposons, T-DNACauses of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous'Spontaneous mutations II:Insertions or deletions caused by replication slippageReplication slippage -> Trinucleotide repeat mutationsReplication slippage -> Trinucleotide repeat mutationsCase: Human Fragile X Mental Retardation syndromeRepeats6-5960-200>200Other example: Huntington’s disease : Poly-glutamine repeats in the CDS -> proteinPhenomenon: “Genetic anticipation”(Transcriptional repression)5'UTRCDS3'UTRCauses of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous’: DNA RepairRepair of bases e.g. Thymidine dimer repairRepair of nucleotides, e.g. Excision repairMeasuring the effects of mutagensAmes testAre mutations directed by the environment?Luria-Delbrück ‘ fluctuation’ experimentLogic: Use the empirical rate of mutation to determine the mutagenicity of a new compound.Measurement of mutation ratesMutation from ad+ to ad- (adenine prototroph to adenine auxotroph)*****Q: Think: The human genome has ~20,000 genes... Ballpark figure for (A+ -> a): = 10-5per gene! My genome may have a new loss-of-function mutation that my parents did not have.Mutation rates per gene 20,000 x 10-5 x 2 = 0.4Mutation rates per basepairMy genome harbors around 180 new point mutations that did not exist in my parent. Q: Think: The human genome has 3 billion basepairs 3 x 10 9 x 3 x 10-8 x 2 = 180How mutagenic is compund X? The Ames testFigure 15-20Strain 1tests for base substitutionsStrain 2tests for indelsIngredients:Salmonella (his - )Potential Mutagen XLiver extract (S9)Medium lacking histidineAmes test - Example: mutagenicity of aflatoxin B1 Figure 15-21TA 100 Straintests for base substitutions(~his-)Strains to testfor indels/frameshiftsAre mutations directed by the environment ?The Luria-Delbrück fluctuation test (1943)E. coli is sensitive to bacteriophage T1.But if one exposes E. coli to phage, a few resistant clones can be found.Sensitive Resistant (R) MutationThe replica plating test E. coli: Demonstrating the presence of T1 phage resistant mutantsTechnique ExperimentTentative conclusion: Mutations preexist in a population, are not generated by exposure to phage.Are mutations directed by the environment ?The Luria-Delbrück fluctuation test (1943)E. coli is sensitive to bacteriophage T1.But if one exposes E. coli to phage, a few resistant cell clones can be found.Sensitive Resistant (R) MutationH1: Mutation to resistance is directed by the phage ('adaptive mutation')H2: Mutation is spontaneous and preexisting in the culture - The phage simply selected for the resistant cells.= Alternative hypothesesLEAP:H2: A pre-existing mutation can have occurred at any time in the past, and the mutants will have proliferated. Predict: No mutation (Most cultures) = no R cells Recent mutation (Some cultures) = few R cells Replicate theMutation long ago (Rare cultures) = many R cells experiment many times!H1: Mutations occur at the same time (upon exposure to phage)Predict: Each replicate experiment should yield ± the same percentage of resistant cells.}“Fluctuation test” hypotheses and predictionsFigure 15-5= Environment directs mutation (False) = Environment does not direct mutations (Correct) H1: Mutation induced by phageH2: Resistant Observationscells (No.)0 101 302 603 304 105 0Resistant Observationscells (No.)0 901 322 163 84 45 2DNA repairConsider: Repair of modified bases. • Direct repair of the defective base(repair of thymidine dimers by photolyase)• Proofreading by DNA polymerase• Excision repair of the defective nucleotides - Base excision (repair deaminated C (uracil)) - Nucleotide excision