L11- DNA repair + Transposable elements

DNA repair-proofreading

• if error occurs during replication..

• DNA pol can reverse and correct error

• 3’-5’ exonuclease activity

MMR

• If proofreading fails, its activated

• mismatches detected, cut, removed

• correct nucleotides inserted by DNA pol

• Msh proteins in humans detect mismtch

• new strand nicked → one that likely has the error, DNA pol and ligase seals nick

Photoreactivation

• Can partially repair UV induced damage

  • PRE enzyme

    • cleaves bond bw pyrimidine dimers

• Requires brief exposure to blue light

• Only some species capable (that can’t remove themselves from light)

  • no animals, some plants

Excision repair

• BER

• NER

• Cut and paste repair

  • Endonucleases recs and cuts error/distortion

  • DNA pol inserts comp nucleotides in missing gap

  • DNA ligase seals final ‘nick’

BER

• corrects DNA containing damaged DNA base (singular damage)

  • deaminations

  • unusual bases (alkylation, oxidation)

• DNA glycosylases detect damage

  • cuts off offending/wrong base

• sugar-p backbone cleaved by endonuclease

• DNA pol inserts correct nucleotide

• Ligase seals the nick

NER

• Repairs bulky lesions that alter/distort double helix

  • Uvr/XP proteins

• Damage due to Pyrimidine dimers, DNA adducts

• Defects w/ NER→ XP, CS, TTD

DS breaks can be caused by ___ and mechanism of repair depends on__

• ionizing radiation

• Stage of cell cycle where damage occurs

HRR

• Repair of ds breaks in late S phase/G2 (sis chromatid made)

• Requires presence of sister chromatid

NHEJ

• Repair of ds DNA breaks in absence of sister chromatid

  • occurs during G1/early S phase

• Take frayed ends + glue together

• Usually results in loss of bases

Ames test

• Test whether a compound can mutate DNA and how strongly

What do you need for Ames test

• ~10^8 salmonella (any bacteria)

  • needs to be auxotrophic (His) meaning have mutation in metabolic pathway so it doesn’t work. Need nutrient supplied to them

• Liver enzyme extract

  • To ensure results of test can be applied to humans

• Add suspected chemical

What happens during Ames test

• Plate mixture→ doesn’t have Hist so bacteria should die but chemical mutagen mutated bacteria in a way that reverses (or suppresses) the pre-existing auxotrophic hist mutation allowing them to survive

• See + result→ lots of colonies bc mutagen allowed them to survive

• Can also see how eff comp mutates DNA using this test bc

How can we see how effective chemical mutagen is using Ames test

• By testing varying concs of chemical.

• Weaker chemical mutagen needs higher dose to get 100 colonies but a stronger one wouldn’t

Mutagenic vs carcinogenic potency

• proportional relationship

• if mutagenic→ causes cancer

How else can we induce mutations in model organisms.. How do we know a mutation has been produced

• chem mutagens (EMS)

• ionizing radiation

• transposable elements

Look for..

• Nutritional mutants (cant make req nutrient)

• visible mutants

• conditional mutants (affected by temps)

• resistance mutants

Detecting nutritional mutants (auxotrophic mutant)

• Grow colonies on complete medium

  • contains all nutrients, including Histidine.

    • Both wild-type bacteria and mutants grow because the nutrient is supplied externally.

• Replica plate onto new media

  • Colonies are transferred in the same spatial pattern to another plate using a sterile pad or velvet.

  • This preserves the exact colony positions.

• Plate lacking the nutrient

  • One replica plate lacks histidine.

  • Wild-type cells grow because they can synthesize histidine themselves.

  • Histidine auxotrophs (mutants with a defective biosynthetic gene) cannot grow.

• Compare plates

  • Compare the original complete plate with the histidine-lacking plate.

  • If a colony appears on the complete plate but is missing on the −histidine plate, that colony is a histidine auxotroph.

• Replica plating→ plate has hist can grow there, plate one without hist

• Plate “mutants” on complete media→ transfer to “lacking media” (non hist, bacteria die)

• Compare initial complete plate to lacking see which colony missing→ isolated auxotrophic bacteria

Transposable elements

• Transposons can insert themselves into various locations w/in genome

• Found in all organisms

  • fx still unkown

  • can disrupt fx of gene by inserting themselves→ mutation

Bacterial Transposons

• IS elements

  • contain only genes needed to mobilize element + insert it into a new location

  • Simplest transposable element

  • inverted repeat seq at ends of sequence

• Tn elements

  • Carry additional genes unrelated to their transposition

    • ex) antibiotic resistance

Ac-Ds system in Maize

• 2 mutations DS (dissociation) and Ac (Activator) are transposable elements in corn

• Movement of Ds gene is dependent on Ac gene

  • Need an Ac nearby for DS to jump

Drosophila transposable elements

• ~5% of genome are mobile elements→ more than 30 families

  • copia + P elements

  • Same as bac (IR at end) but transposase seq has introns and exons

Humans transposable elements

• Humans also have retrotransposons

  • amplify and move within genome through RNA intermediate

  • Initial piece stays there but it’s copied and that copy moves

• Aprox half of human genome is composed of transposable elements

  • most appear to be inactive

Fungal transposable elements

• Heat stress stimulated transposon mobility in human fungal pathogen

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