lecture 20 mutation, repair & recombination ii hchapter 14 hpoint mutations hspontaneous...

LECTURE 20 MUTATION, REPAIR & RECOMBINATION II

chapter 14 point mutations spontaneous mutations biological repair meiotic crossing-over

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...

Meiotic crossing-over is associated

with what process?

LECTURE 20 MUTATION, REPAIR & RECOMBINATION II

DNA repair (1)

double stranded DNA breakage/damage (1)

heteroduplex DNA (½)

recombination (½)

gene conversion (½)

go to schools in person

ask to speak with the guidance councellor

TUTORING CLARK COUNTY HIGH SCHOOL STUDENTS

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 rate = mutation events / gene / time... 1 mutation event (M) 7 cell divisions 1/7 = 0.143

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

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


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


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 lesions depurination = loss of A or G deamination of C U or 5-methyl-C T

depurination > deamination oxidative damage


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 lesions deamination of C U or 5-methyl-C T

GC AT transitions U repairable T not... hot spots


spontaneous lesions oxidative damage of G 8-oxodG (GO)

active O species (O2–, H2O2, OH–)

GC TA transversions

replication errors framshift mutations repeat DNA sequences slipped mispairing


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

INDUCED MUTATIONS

MUTANT 5-BU (transitions)

HA (GC>AT transitions only)

PROFLAVIN (frameshifts)

SPONTANEOUS REVERSION

1 – – – –2 – – + +3 + – – +4 – – – +5 + + – +

1. likely a deletion, perhaps caused by radiation as nothing will revert it2. frameshift, reverted by proflavin and spontaneously3. GC > AT transition, not reverted by 1-way mutagen4. transversion, none of the chemical mutagens will revert it 5. AT > GC transition, reverted by GC > AT transitions only

error-free repair

(a) chemical repair of DNA base damage

BIOLOGICAL REPAIR

(b & c) 2 step process:

1. damaged DNA

deleted,

2. complementary

template strand used

to restore sequence

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 mismatch2. distinguishes incorrect

base from correct errors always on new

unmethylated strand3. 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...

BIOLOGICAL REPAIR

error-prone repair less harmful than no repair at all SOS (already discussed) non-homologous end joining homologous recombination

SOS repair error-prone DNA

polymerases translesion DNA

synthesis mutagenic

BIOLOGICAL REPAIR

non-homologous end joining bind broken ends trimming joining

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

BIOLOGICAL REPAIR

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...


AAAAaaaa

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


AAAaaaaa

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


AAaaaaaa

double-stranded break model of crossing-over


how to think about this problem...


BRANCH MIGRATION ROTATE PERSPECTIVE

BREAKS conversion

“horizontal breakage”


BRANCH MIGRATION ROTATE PERSPECTIVE

BREAKS


recombination

“vertical breakage”


BRANCH MIGRATIONthanks to Bill Engels, Univ. Wisconsin



ROTATE PERSECTIVEthanks to Bill Engels, Univ. Wisconsin



recombination between alleles of a gene intragenic recombination obviously, shorter distances, lower recovery rates

a1 A2+

A1+ a2

GENE A

a1 A2+

a1 a2

A1+ A2+

A1+ a2

unsolved problems (p.478-80) 1- 36

try all of them on your own first then see me / TAs if you have difficulties

SPEND SOME TIME ON...

NEUROBIOLOGY – BIOL 475 / 604

TR 4:00 – 5:15

CBC A108

Behavioral Neurobiology: The Cellular Organization of Natural Behavior by Thomas J. Carew

lecture 20 mutation, repair & recombination ii hchapter 14 hpoint mutations hspontaneous...

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