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LECTURE 20 MUTATION, REPAIR & RECOMBINATION II
chapter 14 point mutations spontaneous mutations biological repair meiotic crossing-over
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LECTURE 20 MUTATION, REPAIR & RECOMBINATION II
you need a piece of paper and a pen or pencil... write your name and student number at the top... give brief answers for the question(s) below... DNA repair (1) double stranded DNA breakage/damage (1) heteroduplex DNA (½) recombination (½) gene conversion (½) Meiotic crossing-over is associated with what process?
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TUTORING CLARK COUNTY HIGH SCHOOL STUDENTS
go to schools in person ask to speak with the guidance councellor Las Vegas High School 6500 E. Sahara Dr. Patrice Johnson,
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SPONTANEOUS MUTATIONS
mutation frequency = mutants / population (0 1) generally rare variable ~ gene
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SPONTANEOUS MUTATIONS
mutation frequency = mutants / population (0 1) = 2/8 = 0.25
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SPONTANEOUS MUTATIONS
mutation frequency = mutants / population (0 1) = 2/8 = 0.25 mutation rate = mutation events / gene / time... 1 mutation event (M) 7 cell divisions 1/7 = 0.143
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SPONTANEOUS MUTATIONS
mutation rate = mutation events / gene / time...
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SPONTANEOUS MUTATIONS
mutation rate = mutation events / gene / time... 1 mutation event (M) 7 cell divisions 1/7 = 0.143 measurement... need # cell divisions = n – initial cell # = 8 – 1 = n in cultures “0 class” frequency
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SPONTANEOUS MUTATIONS
measurement... mutation rate () Poisson distribution mutational events / cell division = mutational events / culture = n frequency of cultures with 0 mutants = e–n e.g. if n = 0.2 108 cells 0 class = e–n = 11/20 = 0.55 0.55 = e–(0.2 108) 3 10–8 events / cell division
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SPONTANEOUS MUTATIONS
Calculate the mutation rate / cell division / resistance gene in 108 Tons E. coli spread on 100 minimal media plates with T1 phage where 31 of these plates had no growth. n = 108 cells (–1 = # cell divisions) 0 class = e–n = 31/100 = 0.31 0.31 = e–(108) = 1.17 10–8 events / cell division / gene
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SPONTANEOUS MUTATIONS
spontaneous lesions depurination = loss of A or G deamination of C U or 5-methyl-C T depurination > deamination oxidative damage
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SPONTANEOUS MUTATIONS
spontaneous lesions depurination = loss of A or G break sugar • base glycosidic bond mammalian cells loose ~ 104 purines / cell / gen. error-prone SOS repair
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SPONTANEOUS MUTATIONS
spontaneous lesions deamination of C U or 5-methyl-C T GC AT transitions U repairable T not... hot spots
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SPONTANEOUS MUTATIONS
spontaneous lesions oxidative damage of G 8-oxodG (GO) active O species (O2–, H2O2, OH–) GC TA transversions
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SPONTANEOUS MUTATIONS
replication errors framshift mutations repeat DNA sequences slipped mispairing
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INDUCED MUTATIONS
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INDUCED MUTATIONS reversion tests
tell us about the nature of the forward mutation ... and action of the mutagens used e.g. mutagen specificity implied if it does not revert its own forward reaction
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INDUCED MUTATIONS reversion tests... example question, p. 479, # 27 1
MUTANT 5-BU (transitions) HA (GC>AT transitions only) PROFLAVIN (frameshifts) SPONTANEOUS REVERSION 1 – 2 + 3 4 5 likely a deletion, perhaps caused by radiation as nothing will revert it frameshift, reverted by proflavin and spontaneously GC > AT transition, not reverted by 1-way mutagen transversion, none of the chemical mutagens will revert it AT > GC transition, reverted by GC > AT transitions only
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BIOLOGICAL REPAIR error-free repair
(a) chemical repair of DNA base damage (b & c) 2 step process: 1. damaged DNA deleted, 2. complementary template strand used to restore sequence
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BIOLOGICAL REPAIR chemical repair of DNA base damage (a)
photorepair with photolyase + visible light
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BIOLOGICAL REPAIR chemical repair of DNA base damage (a)
alkyltransferase (e.g. methyltransferase) removes methyl groups, e.g. added by EMS
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BIOLOGICAL REPAIR homology-dependent repair, 2 general types
excision repair (b) base excision repair nucleotide excision repair in prokaryotes transcription-coupled repair in eukaryotes postreplication repair (c) in prokaryotes in eukaryotes
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BIOLOGICAL REPAIR excision repair base excision repair
DNA glycosylases cleave base-sugar bonds (different types) apurinic or apyrimidinic sites enzymes: 1. AP endonuclease 2. excision exonuclease DNA polymerase, 4. ligase more ways to damage bases than # of DNA glycosylases...
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BIOLOGICAL REPAIR excision repair
nucleotide excision repair (prokaryotes) detects distortions in DNA enzymes: 1. uvrABC exinucleases removes 8+4 nucleotides 2. DNA pol I 3. ligase
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BIOLOGICAL REPAIR excision repair
transcription-coupled repair (eukaryotes) important, many cells terminally differentiated & no longer dividing no replication-repair damage blocks transcription detects distortions in DNA
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BIOLOGICAL REPAIR excision repair
transcription-coupled repair (eukaryotes) repairisome (>20 subunits) 7 of these part of transcription machinery removes ~ 30 nucleotides preferentially repairs template strand bubble forms, excision, DNA synthesis & ligation
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BIOLOGICAL REPAIR excision repair postreplication-repair (prokaryotes)
replication errors missed by proof-reading function of DNA pol mismatch-repair system 1. recognizes mismatch 2. distinguishes incorrect base from correct errors always on new unmethylated strand 3. excise incorrect base repair synthesis
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BIOLOGICAL REPAIR excision repair postreplication-repair (eukaryotes)
microsatelites, some in critical coding regions slipped-mispairing replication errors missed by proof-reading function of DNA pol mismatch-repair system
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BIOLOGICAL REPAIR error-prone repair double stranded breaks from
reactive oxygen species ionizing radiation (X-rays, -rays) unlike single stranded damage no exact template, no error-free repair... error-prone repair less harmful than no repair at all SOS (already discussed) non-homologous end joining homologous recombination
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BIOLOGICAL REPAIR SOS repair error-prone DNA polymerases
translesion DNA synthesis mutagenic
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BIOLOGICAL REPAIR non-homologous end joining bind broken ends trimming
involved in generating rearrangements of antibody genes in mammalian immune systems
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BIOLOGICAL REPAIR homologous recombination
homologous sister chromatids trim ends DNA-protein filament homology search & strand invasion DNA synthesis ligation ~ crossing-over
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MEIOTIC CROSSING-OVER
initiated by double-stranded chromosome breakage between 2 homologous non-sister chromatids no gain or loss of genetic material 2 steps double stranded breakage heteroduplex DNA formed, derived from non-sister chromatids on homologous chromosomes
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MEIOTIC CROSSING-OVER
evidence first from aberrant ratios observed in fungi aberrant asci have > 4 copies of on genotype extra copies changed through gene conversion 5:3 ratio from non-identical sister spores in meiosis with heteroduplex... A a
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MEIOTIC CROSSING-OVER
evidence first from aberrant ratios observed in fungi aberrant asci have > 4 copies of on genotype extra copies changed through gene conversion 5:3 ratio from non-identical sister spores in meiosis with heteroduplex not repaired A a
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MEIOTIC CROSSING-OVER
evidence first from aberrant ratios observed in fungi aberrant asci have > 4 copies of on genotype extra copies changed through gene conversion 6:2 ratio from non-identical sister spores in meiosis with heteroduplex repaired A a
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MEIOTIC CROSSING-OVER
double-stranded break model of crossing-over
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MEIOTIC CROSSING-OVER
double-stranded break model of crossing-over
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MEIOTIC CROSSING-OVER
double-stranded break model of crossing-over
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MEIOTIC CROSSING-OVER
how to think about this problem... ROTATE PERSPECTIVE BRANCH MIGRATION conversion “horizontal breakage” BREAKS
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MEIOTIC CROSSING-OVER
how to think about this problem... BRANCH MIGRATION ROTATE PERSPECTIVE BREAKS recombination “vertical breakage”
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MEIOTIC CROSSING-OVER
how to think about this problem... BRANCH MIGRATION thanks to Bill Engels, Univ. Wisconsin
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MEIOTIC CROSSING-OVER
how to think about this problem... ROTATE PERSECTIVE thanks to Bill Engels, Univ. Wisconsin
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MEIOTIC CROSSING-OVER
recombination between alleles of a gene intragenic recombination obviously, shorter distances, lower recovery rates a1 A2+ a1 a2 A1+ A2+ A1+ a2 a A2+ A a2 GENE A
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SPEND SOME TIME ON... unsolved problems (p.478-80) 1- 36
try all of them on your own first then see me / TAs if you have difficulties
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NEUROBIOLOGY – BIOL 475 / 604 TR 4:00 – 5:15 CBC A108
Behavioral Neurobiology: The Cellular Organization of Natural Behavior by Thomas J. Carew
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