Chapter 14 posted.pptx

Mutation, transposition, and repair Outline • Mutations • How they happen • Transposition • How mutations are repaired • A genetic screen General mutation classifications • How mutation happens: – Spontaneous mutation – Induced mutation • Cells affected: – Somatic mutation – Germline mutation...

Mutation, transposition, and repair Outline • Mutations • How they happen • Transposition • How mutations are repaired • A genetic screen General mutation classifications • How mutation happens: – Spontaneous mutation – Induced mutation • Cells affected: – Somatic mutation – Germline mutation • Affected chromosome – Autosomal mutation – X-linked mutation Classification: molecular change • Point mutation/base substitution: – Type of mutation: • Transition: – pyrimidine to pyrimidine or purine to purine • Transversion: – purine to pyrimidine or pyrimidine to purine – Result of mutation: • Missense mutation: changes the amino acid • Nonsense mutation: changes amino acid to STOP • Silent mutation: No change in amino acid • Frameshift mutation – Changes reading frame, altering all amino acids after the mutation • Regulatory mutation Classification: phenotypic change • Loss-of-function/Gain-of-function – Complete loss-of-function: Null mutation • Visible mutations • Nutritional mutation – Prototroph to auxotroph • Biochemical mutation – Sickle cell anemia, PKU Classification: phenotypic change • Behavioral mutation • Lethal mutation • Conditional mutation – Temperature sensitive mutation What causes mutations? • Errors in replication • DNA damage: – Double strand breaks, when do we know these occur? • • • • Recombination Translocation Replication (Gyrase) Transposition • Mutagens – Includes chemicals and naturally occurring substances that cause mutation Replication errors • Despite 3’-5’ exonuclease (proofreading) activity, DNA polymerases still make errors. • Replication slippage: replication errors that occur at highly repetitive sequences – Huntington disease (HD) and the poly glutamine tract… – CAG repeat (codes glutamine) >36 glutamines = HD http://www.web-books.com/MoBio/Free/Ch7F3.htm Tautomeric shifts 14-2 14-3 Naturally occurring mutations: Depurination/Deamination • Depurination: loss of a base in the intact double helix (usually purine) – If not repaired, random base inserted • Deamination: conversion of amino group on cytosine or adenine to keto group – Alters the base-pairing specificity: 14-4 Induced mutations: DNA damage from chemicals • Base analogs: Structurally similar, but can change base pairing specificity via increase chance of tautomeric shift. – Example: 5-BU (5bromouracil) is similar to thymine (T) but can pair with A or G 14-5 Induced mutations: DNA damage from chemicals • Alkylating agents: Alkylation changes the base pairing specificity of a nucleotide – Example: Ethyl-methane sulfonate (EMS): Causes G to pair with T instead of C • Acridine dyes: Intercalate DNA (insert between purines and pyrimidines) causing deletions or insertions – Example: acridine orange 14-6 Induced mutations: DNA damage from irradiation Visible spectrum • UV light • Ionizing radiation 14-7, 14-8 Transposable elements • Two general varieties – Cut-and-paste transposons – Copy-and-paste transposons 9-40 nucleotides long • Requirements – Transposable element: DNA sequence – Transposase: enzyme responsible for mobilization • Can contain other genes – Drug resistance, selectable markers, gene to be studied… 14-16 Transposable elements • Ac & Ds in corn – Barbara McClintock – Ac: contains transposase – Ds: does not contain transposase, depends on Ac for movement • P elements in Drosophila – Transposase expressed specifically in germline – Germline transformation = transgenics • Transposable elements are mutagenic 14-17 Exploiting transposable elements DNA repair • • • • • • Proofreading Mismatch repair Post replication repair Photoreactivation repair Excision repair Double-strand break repair DNA repair: Proofreading • DNA polymerase III error rate: – 1 error in 100,000 bases • 3’-5’ exonuclease activity, improves this rate 100 fold: – 1 error in 10,000,000 bases • How many basepairs are replicated (Human genome)? • Haploid genome = ~3 billion base pairs • Diploid genome = ~6 billion base pairs • Total: ~12 billion bases • ~1200 “uncorrected” errors per cell, each time it replicates! – FYI: There are trillions of cells in the human body • Must have other mechanism(s) to repair errors! Mismatch repair • Base-pair mismatches are detected and repaired • Which strand is the correct one? – DNA methylation: Which is correct base? • Adenine methylase trails the polymerase during replication • newly synthesized strands are temporarily unmethylated • The repair: – Endonuclease nicks the DNA strand to be repaired (either 3’ or 5’ of mismatch) – Exonuclease degrades the DNA until the mismatch is reached – Gap is filled by DNA polymerase and “sealed” by DNA ligase 14.3 Post replication repair Note: repair only fixes the “new” strand, lesion remains on parental strand 14-9 Photoreactivation repair • PRE: Photoreactivation enzyme – Activated by light in the blue range of the visible spectrum – Requires absorption of a light photon in order to cleave the dimer • Not found in humans 14-13 Base excision repair (BER ) • Mismatch distorts the helix structure • Note: Glycosylases are specific for each nucleotide – Uracil – Alkylated bases – Deaminated bases 14-10 Nucleotide excision repair (NER ) • Repairs “bulky” lesions (e.g. pyrimidine dimers) – Recognized by uvr gene products 1. Nuclease excises the lesion 2. DNA polymerase I fills the gap 3. DNA ligase seals the gap 14-11 Double-strand break (DSB) repair • DSB detected and digested (5’-3’ exonuclease) • Sister chromatid provides homologous template for repair 14-13 DSB repair movie Special thanks to Dr. Steve White (St. Jude) Ames test: detects mutagenic substances Why liver enzymes? 14-14 • Reversion: mutation back to WT Use of mutation • Geneticists will induce mutation in model organisms to study gene function – Radiation – Chemicals – Transposable elements • But mutation is random! • Genetic screens are used to identify mutations of interest – Phenotype • How do scientists detect recessive mutations? – answer: get creative! WT bcd - Ligand P P P • SOCS = suppressor of cytokine signaling – Negatively regulate JAK-STAT pathway • Drosophila genome contains 3 SOCS genes – 2 are on second chromosome: • socs36E, socs44A P P STAT STAT P STAT STAT P JAK P STAT STAT P RECEPTOR JAK RECEPTOR P JAK-STAT Pathway STAT responsive genes, including SOCS EMS screen overview • Exposure to EMS mutagen • Establishment of balanced lines • Genetics: – Test crosses - screen – Complementation analyses • Sequence DNA Factors to remember • We don’t know the mutant phenotype • Mutation is likely recessive so we won’t see it in flies that are heterozygous • Mutation can be lethal – Homozygotes die as embryos • We must be able to follow which chromosome our mutation is on from generation to generation – We must prevent recombination: • Male Drosophila do not undergo recombination! • Use of balancer chromosomes EMS Screen Scheme 600 cn bw sp * X cn bw sp * cn bw sp * Sco Sco CyO cn bw sp * CyO • Sco (Scutoid) is a dominant marker resulting in loss of hairs on the scutelum • CyO (Curly-O) is a balancer chromosome, it contains: • a dominant marker (Curly) causing the wings to curl upward • a series of nested inversions How does CyO prevent recombination? 7477 cn bw sp * CyO cn bw sp * Sco cn bw sp * CyO X Sco CyO X (sibs) Sco CyO CyO CyO cn bw sp * CyO 3986 BALANCED STOCKS Deficiency map (Socs44A region) NCX10 cn9 Drlrv18 cn83c CA53 42E 43 44A 44B socs44A 42A 44C 44D 44A3 – 44E 45 Test crosses cn bw sp * CyO X Deficiency CyO cn bw sp * Deficiency cn bw sp * CyO CyO Deficiency CyO CyO (anything other than wildtype) Summary of crosses Stage # of crosses # socs44A candidates Establish mutant stocks 20,000 3986 Test crosses 4,000 155 Secondary screens 1,000 43 Total 25,000 socs44A Complementation analysis 42 42 265 565 680 705 754 1014 1080 1547 1679 1824 1938 1997 2004 2008 2014 2589 2608 2933 3488 265 WV 565 PCVmod v 680 WV L v 705 v v v v 754 v SL(4/40) v v v 1014 WV v v v v v 1080 PCVmod v v v SL(2/30) v v 1547 v v v v v v v v 1679 v v v v v v v v SL(6/30) Complementation analysis: • 23 complementation groups • Largest group has 9 members • 9 groups have 3 members • Rest are “singles” 1824 v L v L v v v v v v 1938 v v v v v v v v v v v 1997 v v v v v v v v v v v v 2004 v v v v v SL(2/25) v v v v v v v 2008 v v v v v v v v v v v v v v 2014 2589 2608 2933 SL(10/70) PCV v v v L v L v v L v v L v L v v v v v v SL(8/40) v v v v v SL(1/30) SL(3/40) L L v v SL(3/20) v v v v v v L SL(5/30) L v v v v v v v v v v v v v v SL(4/50) v v L v v SL(3/60) v Summary of crosses Stage # of crosses # socs44A candidates Establish mutant stocks 20,000 3986 Test crosses 4,000 155 Secondary screens 1,000 43 Complementation analysis 1,600 23 Total 26,600 Sequencing • Viable mutants can be sequenced directly • Recessive lethal mutants are balanced over CyO,GFP • Select non-GFP embryos/larvae from each complementation group for sequencing • Assemble sequences and compare to original cn bw sp chromosome to discover precise mutation EMS screen summary • Exposure to EMS mutagen • Establishment of balanced lines • Genetics: – Test crosses - screen – Complementation analyses • Sequence DNA to determine precise mutation Review • Mutations • How they happen • Transposition • How mutations are repaired • A genetic screen