LECTURE 20 MUTATION, REPAIR & RECOMBINATION II chapter 14 point mutations spontaneous mutations biological repair meiotic crossing-over

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?

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, Principal @ 799.0180

SPONTANEOUS MUTATIONS mutation frequency = mutants / population (0  1) generally rare variable ~ gene

SPONTANEOUS MUTATIONS mutation frequency = mutants / population (0  1) = 2/8 = 0.25 

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       

SPONTANEOUS MUTATIONS mutation rate = mutation events / gene / time...

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 = 7  n in cultures “0 class” frequency       

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

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

SPONTANEOUS MUTATIONS spontaneous lesions depurination = loss of A or G deamination of C  U or 5-methyl-C  T depurination > deamination oxidative damage

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

SPONTANEOUS MUTATIONS spontaneous lesions deamination of C  U or 5-methyl-C  T GC  AT transitions U repairable T not... hot spots

SPONTANEOUS MUTATIONS spontaneous lesions oxidative damage of G  8-oxodG (GO) active O species (O2–, H2O2, OH–) GC  TA transversions

SPONTANEOUS MUTATIONS replication errors  framshift mutations repeat DNA sequences  slipped mispairing

INDUCED MUTATIONS

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

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

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

BIOLOGICAL REPAIR chemical repair of DNA base damage (a) photorepair with photolyase + visible light

BIOLOGICAL REPAIR chemical repair of DNA base damage (a) alkyltransferase (e.g. methyltransferase) removes methyl groups, e.g. added by EMS

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

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 3. DNA polymerase, 4. ligase more ways to damage bases than # of DNA glycosylases...

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

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

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

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

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

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

BIOLOGICAL REPAIR SOS repair error-prone DNA polymerases translesion DNA synthesis mutagenic

BIOLOGICAL REPAIR non-homologous end joining bind broken ends trimming involved in generating rearrangements of antibody genes in mammalian immune systems

BIOLOGICAL REPAIR homologous recombination homologous sister chromatids trim ends DNA-protein filament homology search & strand invasion DNA synthesis ligation ~ crossing-over

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

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

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

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

MEIOTIC CROSSING-OVER double-stranded break model of crossing-over

MEIOTIC CROSSING-OVER double-stranded break model of crossing-over

MEIOTIC CROSSING-OVER double-stranded break model of crossing-over

MEIOTIC CROSSING-OVER how to think about this problem... ROTATE PERSPECTIVE BRANCH MIGRATION conversion “horizontal breakage” BREAKS

MEIOTIC CROSSING-OVER how to think about this problem... BRANCH MIGRATION ROTATE PERSPECTIVE BREAKS recombination “vertical breakage”

MEIOTIC CROSSING-OVER how to think about this problem... BRANCH MIGRATION thanks to Bill Engels, Univ. Wisconsin

MEIOTIC CROSSING-OVER how to think about this problem... ROTATE PERSECTIVE thanks to Bill Engels, Univ. Wisconsin

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  a1 A2+ A1+ a2 GENE A

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

NEUROBIOLOGY – BIOL 475 / 604 TR 4:00 – 5:15 CBC A108 Behavioral Neurobiology: The Cellular Organization of Natural Behavior by Thomas J. Carew