Lecture 8 (biol3600) dna damage and repair - winter 2012


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Lecture 8 (biol3600) dna damage and repair - winter 2012

  1. 1. http://www.youtube.com/watch?v=bSRC1_OZPIgGo 1:30minhttp://www.youtube.com/watch?v=4fCCVU4y7oEMust see 1:15min DNA mutation and repair Assigned reading: Chapter 18
  2. 2. Mutations What are they?• Mutation - Any change made to the DNA sequence or chromosome structure - Important general properties: 1) Not inherently good or bad - Can lead to disease/death, but can also create new alleles (evolution) - Example: HbS, sickle-cell anemia - Can be w/in junk (usually), coding sequence, promoters 2) They are permanent – can’t be removed or repaired (damage vs mutation) 3) They do not selectively occur (random) - Do Antibiotics CAUSE Antibioticsr mutations? NO!! - Mutation occurs randomly  Antibiotic kills all non-mutants  survivors Abicr• Mutations are classified in different ways: 1) Size - Mutations can either involve large portions of chromosomes or small regions a) Chromosomal mutations – Large segments of chromosomes are deleted, inverted, moved, or duplicated (DISCUSSED LATER) b) Gene mutations – Smaller changes in the DNA sequence - One or a few nucleotides
  3. 3. Mutations Classification18.1) Mutations are Inherited Alterations in theDNA Sequence• Mutations are classified in different ways: 1) What causes them? (more later) - Some due to natural biochemical events - Called spontaneous mutations - Others helped along by some artificial factor (chemicals, radiation, viral) - Called induced mutations 2) Type of cell that contains mutated DNA: - Somatic mutations = Arise in the DNA of somatic cells (normal diploid) - NEVER passed onto the next generation - Mutating cells in your arm  Won’t go to kids - Earlier in development  Worse outcomes (Why?) - Germ-line mutations = Mutations arise in the DNA of gamete-forming tissue (those cells that produce sperm and eggs) - Transmitted to the offspring  Pass onto future generations
  4. 4. Mutations Classification• Mutations are classified in different ways: 3) Other classes of mutations a) Some are incompatible with life (lethal mutations) - If lead to prenatal death  embyronic lethal mutations b) Some only produce an effect under certain environmental conditions such as hot temperatures (conditional mutations) - Condition doesn’t cause it, but allows it to be expressed c) Some reverse (suppress) the effect of a previous mutation (suppressor mutations) - Not a direct reversal of the mutation - Intragenic – 2nd mutation in the same gene - Mutation 1 alters protein structure, 2 alters it back - Intergenic – 2nd mutation in totally different gene - Usually seen in pathways or protein multimers - Mutant protein 1 is defective, mutant protein 2 does the job of protein 1
  5. 5. Different types of gene mutations Base-pair substitutions• Small gene mutations come in 3 main varieties: 1) Base-pair substitutions - One nucleotide is changed to a different nucleotide - Possible outcomes on the amino acid sequence : 1) No effect– usually see this if the 3rd nucleotide of a codon is changed - Called a silent mutation 2) Change causes the wrong amino acid to be inserted - Called a missense mutation - If new type of aa, changes protein shape - If similar aa, may have little effect (neutral) - Ex: Sickle-cell, cystic fibrosis, Tay-Sachs 3) Change turns the codon into a stop codon - Causes the polypeptide to stop growing (protein will be truncated/shorter) - Called a nonsense mutation
  6. 6. Alterations can happen with Autosomal Chromosomes Overview and some terms Cells end up with extra chromosome Cells end up with extra sets of chromosomes
  7. 7. Alterations can happen with Autosomal Chromosomes Overview and some terms• Cells can acquire larger alterations in chromosome number or structure - These are more often harmful (as with the sex chromosomes)• Alterations in chromosome number - General terms: 1) Aneuploidy – An organism gains or loses 1 or more chromosomes (but not a full set) - Monosomy – Individual lacks 1 chromosome (example: XO has 45 chr.) - Trisomy – Individual has 1 extra chr. (example: XXY has 47 chr.) - Tetrasomy – 2 extra, penta..... 2) Polyploidy – An organism has 1 or more extra SETS of chromosomes - Diploidy – normal (2n) - Triploidy – 1 extra set (3n), tetraploidy (4n)
  8. 8. Alterations in chromosome number Polyploidy• Polyploidy - Not very common in the animal world (some fish and amphibians), but plants commonly have extra sets of chromosomes• Organisms acquire an extra set of chromosomes in 2 major ways: 1) Autopolyploidy – Occurs when an organism acquires an extra copy of its own chromosomes - AA  AAA (tri), AAAA (tetra), etc.... 2) Allopolyploidy – Occurs when chromosome sets from 2 different species are combined - AA + BB  AABB• Autopolyploidy - Autotriploids (AAA) can be generated in 3 major ways: 1) During meiosis, one gamete (of a diploid) receives both copies of every chr (AA).If that gamete then gets fertilized by a haploid gamete (A)  result AAA 2) One egg is fertilized by 2 sperms 3) If a (normal) tetraploid organism mates with a (normal) diploid individual - Tetraploid gametes would be AA, diploid gametes A  result AAA
  9. 9. Alterations in chromosome number Polyploidy• Autopolyploidy - Autotetraploids (AAAA) arise from the premature stoppage of mitosis (in early embryos)  A cell will end up with twice the normal number of chromosomes - Most autopolyploids are usually sterile - Produce bad gametes b/c of abnormal chr. separation during meiosis - Examples: Seedless watermelons• Allopolyploidy - This occurs when 2 haploid gametes of 2 closely related species accidentally fuse - A + B  AB - These initial individuals would be sterile because A and B chr. can not synapse during prophase (can not separate properly) - Occasionally, these organisms will undergo a chromosomal doubling at early stage - AB  AABB (called allotetraploid)
  10. 10. Alterations in chromosome number Polyploidy• Allopolyploidy - Can see hybrid allotetraploid plants in nature (never in animals – mating problems) - They are fertile b/c they have 2 good copies of each chromosome (meiosis occurs normally) - These plants often have traits from both species - Can be very economically important if the resulting allotetraploid express good traits from each species - Scientists attempt to engineer allopolyploids with the hopes of making lots of $$$$$
  11. 11. Alterations in chromosome number Aneuploidy  Trisomy• Trisomy is also a very bad situation for animals and plants - Animals that have an extra sex chromosome are usually viable, but have various developmental defects - Animals that have an extra autosome are usually not viable - Only a few examples of viable trisomic conditions involving autosomes - The smaller the chromosome, the likelier that the organism can tolerate having an extra one - Plants are a little more tolerant of extra autosomes as far as viability goes - Having extra autosomes usually alters the phenotype• Examples of autosomal trisomic conditions in humans 1) Down syndrome (trisomy 21) - Most common trisomic condition (almost all others are embryonic lethal) - Although individual phenotypes vary, most are short and have mental impairment, heart/lung malformations, and abnormal skeletal development
  12. 12. Alterations in chromosome number Aneuploidy  Trisomy• Examples of autosomal trisomic conditions in humans 1) Down syndrome (trisomy 21) - Most cases are due to nondisjunction during meiosis I - 95% of the cases are due to nondisjunction in the moms egg - Incidence of Down syndrome directly correlates with moms age (no one really knows why) 2) Patau syndrome (trisomy 13) - Infants have severe developmental problems in almost all organ systems and usually, when born alive, only survive a few months. - Older the mom and dad, greater chance of having a baby with PS 3) Edward syndrome (trisomy 18) - Same as in PS, except most infants that have this syndrome are femalehttp://www.youtube.com/user/paulawaziry?feature=mhee#p/c/C038F6E6BFE2738A/49/lP6aLp-4zg0
  13. 13. • Types of Chromosomal Mutations:1) Change in the number of chromosomes (what we have seen so far)2) Rearrangement of genes - Deletions - Inversions - Duplications - Translocations Reciprocal Non-reciprocal
  14. 14. Alterations in chromosome structure• Changes in chromosomal structure and arrangement are usually due to the introduction of a chromosomal break (spontaneous or induced by chemicals/ radiation), followed by the loss or rearrangement of the chr. pieces• When chromosomes break, the free ends are "sticky" and will rejoin with other free ends in a nonspecific way• The major types of chromosomal rearrangements include:
  15. 15. • Types of Chromosomal Mutations:Rearrangement of genes
  16. 16. Alterations in chromosome number• There is an example of partial monosomy in humans - These individuals are missing a significant portion of the small arm of chr. 5 - Individuals with this deletion suffer from a condition called Cri-du-chat syndrome ("cry of the cat")• Cri-du-chat symptoms - Individuals are usually mentally handicapped and usually suffer some physical abnormalities - Heart and GI tract are usually malformed - Malformation of the larynx and glottis leads infants to have a characteristic cry that sounds like a cats meowhttp://www.youtube.com/user/paulawaziry?feature=mhee#p/c/C038F6E6BFE2738A/19/Bf3O_Q31ZUg
  17. 17. • Types of Chromosomal Mutations:Rearrangement of genes:Duplication
  18. 18. Alterations in chromosome structure Deletions• A deletion is the loss of a portion of a chromosome - Usually bad (cri-du-chat), but can be good• Breaks can occur at different places within the chromosome - A single break can give rise to a terminal deletion - Two internal breaks can give rise to a intercalary deletion• When a chromosome breaks and the pieces remain apart, what determines which piece will be lost?  Whatever piece has the centromere will be retained by the cell (mitosis)• Chromosomes containing deletions will usually have normal counterpart - How do they synapse properly during meiosis (remember that chr. match up perfectly during prophase I)? - When one homolog is missing a piece, the other one will loop out the "extra" sequence so that they can synapse properly
  19. 19. • Types of Chromosomal Mutations:Rearrangement of genes:Ex: Huntington disease
  20. 20. Alterations in chromosome structure Duplications• A duplication is a when any piece of a chromosome (can be a single gene) is present more than once in the genome• How do duplications occur? 1) Improper crossing over between homologous chromosomes during prophase I - Two nonsister chromatids should exchange the exact same genes - Occasionally there is an error and one chromatid receives too much and the other receives too little 2) An error occurs during DNA replication that leads to the same piece being added twice• Gene duplications can be beneficial for organisms 1) In some cases, cells containing chr. duplications will be able to grow better than their normal counterpart - Example: Most organisms have multiple copies of each type of rRNA gene - More copies of rRNA gene, more rRNA produced, more ribosomes, more protein
  21. 21. mutation: any heritable change in the genetic material (excludes changes caused by normal recombination events) C--T happens The integrity of genomic DNA is constantly under threat, even inperfectly healthy cells. DNA damage can result from the action ofendogenous reactive oxygen species, or from stochastic errors in replication or recombination, as well as from environmental and therapeutic genotoxins.
  22. 22. Spontaneous mutations: a mutation that occurs inthe absence of known mutagens uncorrected errors that occur during DNA replication, repair or recombination spontaneous lesions that occur to the DNAmolecule under normal physiological conditions andthat are not repaired by the cell’s DNA excision repairprocesses
  23. 23. Alterations in chromosome structure Duplications• Gene duplications can be beneficial for organisms 2) Encourage the creation of new genes  Evolution - Evolution generally requires: a) Formation of new alleles of existing genes (protein still has the same general function) OR b) Formation of brand new genes that encode proteins with novel functions - Suggestion: Take that essential gene, duplicate it, and mutate the duplicate until it is a brand new gene (that encodes a different protein) - You now have the essential "old" gene and a brand new gene  These gene duplication events are thought to be a major source of new genes and a driving force in evolutionary change - Examples: Hemoglobin and myoglobin Trypsin and chymotrypsin
  24. 24. • Types of Chromosomal Mutations:Rearrangement of genes
  25. 25. Alterations in chromosome structure Inversions• Inversion – A piece of the chromosome gets inverted 180º within the chromosome• How does an inversion take place? - A double chromosomal break occurs - Based on where the pieces are positioned with respect to one another, they may be ligated in the wrong place• Inversions can be classified based on the appearance of the arms after the event - Paracentric – Both breaks occur within 1 arm. Centromere is not involved in the inversion. Arm ratios remain unchanged - Pericentric – Breaks occur in each arm. Arm ratios will be changed following ligation• Inversions are a problem for gamete formation and new positions may be bad! WHY?
  26. 26. • Types of Chromosomal Mutations:Rearrangement of genes:Translocation involves 2 chromosomesHere is a non-reciprocal translocation- involves non-homologous pairs
  27. 27. Alterations in chromosome structure Translocations• Translocation – A segment of a chromosome is transferred onto a nonhomologous chromosome - If 2 nonhomologous chr. trade random pieces  reciprocal translocation - If 1 chr. just takes a piece from another  nonreciprocal translocation• How does this occur? - It they switch end segments, just need 2 chr. to randomly be close together and each have a break
  28. 28. Alterations in chromosome structure Translocations• Translocations are like inversions in thatno genetic info is lost in the process - The info is just put in a different place - Often has no real effect on organism viability• When do translocations (and inversions) create problems: 1) The region of the chromosome near the centromere is transcriptionally inactive - If a translocation ends up moving a highly transcribed gene near the centromere, it will now be shut down 2) Promoters tightly regulate the rate of transcription for each specific gene - If translocation ends up moving a gene to a new location so that it is under the control of a different promoter, that can be very bad - What if a cell death gene that is only activated when the cell is in trouble is moved downstream of a constitutive promoter? - translocations can lead to cancer
  29. 29. Different types of gene mutations Insertions/deletions• Small gene mutations come in 3 main varieties: 2) Insertions/deletions – An extra nucleotide gets added or removed - VERY BAD b/c it causes a frameshift (shift in the reading frame) - All amino acids after ins/del will be wrong!! THE DOG BIT THE MAN THE DOG C BIT THE MAN. delete the G add an extra C THE DOB ITT HEM AN THE DOG CBI TTH EMA N mRNA PARENTAL DNA amino acid sequence ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE altered mRNA BASE INSERTION altered amino acid sequence ARGININE GLYCINE LEUCINE LEUCINE GLUTAMATE
  30. 30. Phenotypic Effects of Mutations• Forward mutation: wild type  mutant type• Reverse mutation: mutant type  wild type• Missense mutation: amino aciddifferent amino acid• Nonsense mutation: sense codon nonsense codon• Silent mutation: codonsynonymous codon• Neutral mutation: no change in function
  31. 31. Different types of gene mutations Expansion of repeats• Small gene mutations come in 3 main varieties: 3) Expansion of trinucleotide repeats (TNRE) - Some loci contain a series of trinucleotide repeats (e.g. CAGCAGCAG...) next to a gene or inside the gene - Everyone has them – usually stable copy numberhttp://www.youtube.com/watch?v=Symw0nU7Hys - Abnormal event can occur  Copy number increases - Ex: Normally have 10 copies of CAG on chr. 8  inc. to 200 copies - What causes the increase? NO ONE REALLY KNOWS - Abnormal DNA structure causes DNA pol to slip and copy section 2x - Such expansion often leads to disease - If in a gene, expansion increases # of a.a. - If next to a gene, can trigger methylation of gene - TNRE disorders usually get worse each generation - Expansion grows  worse symptoms - Called anticipation
  32. 32. Causes of mutations Damage becomes mutation• How does DNA damage get converted into permanent mutations?Common theme :1) A change occurs in the structure of a nt (lesion/damage)2) DNA replication occurs – DNA pol puts "wrong" nt across from the lesion3) 2nd DNA rep occurs – Wrong nt serves as a template for complimentary wrong ntRESULT: DNA now contains a completely wrong PAIR  cell sees as normal!! lesion
  33. 33. Causes of DNA damage Spontaneous damage• Causes of spontaneous damage include: 1) Errors of DNA polymerase - Polymerases and proofreading/repair enzymes are not perfect - Some major causes of spontaneous errors during replication include: a) Strand slippage (see TNRE) - Repeats cause abnormal loop  DNA pol copies same thing 2x b) Defective proofreading
  34. 34. Causes of DNA damage Spontaneous damage• Causes of spontaneous damage include: 2) Tautomeric shifts - Nitrogenous bases can exist in different chemical forms called structural isomers - "Normal" forms  A-T, C-G bonding - "Rare" isomers  Abnormal base pairing - Ex: Abnormal T prefers to H bonds w/ G - Conversion between normal and abnormal isomers occurs naturally at some low rate - VERY BAD if it occurs right before DNA replication - DNA pol will read rare form and insert the wrong base across (common theme discussed) - Not a major source of mutations
  35. 35. Concept Check 1Which of the following changes is a transition base substitution? a. Adenine is replaced by thymine. b. Cytosine is replaced by adenine. c. Guanine is replaced by adenine. d. Three nucleotide pairs are inserted into DNA.
  36. 36. Concept Check 1Which of the following changes is a transition base substitution?a. Adenine is replaced by thymine.b. Cytosine is replaced by adenine.c. Guanine is replaced by adenine.d. Three nucleotide pairs are inserted into DNA.
  37. 37. Causes of DNA damage Spontaneous damage• Causes of spontaneous damage include: 2) Tautomeric shifts (common theme again) a) A nitrogenous base shifts from the common tautomer to the rare version (called tautomeric shift) – let’s use a rare “A” tautomer b) DNA replication begins - One strand is normal - When DNA pol sees the rare “A” tautomer in the template , it will insert a C into the new strand  Assume the tautomer goes back to the normal form c) DNA replication begins again - "Wrong" C serves as a template for a G - A permanent (unrepairable) mutation has occurred  Shift, DNA rep I (wrong base put in), DNA rep II (wrong base is template)
  38. 38. Causes of DNA damage Spontaneous damage• Causes of spontaneous damage include: 3) Depurination and deamination - Depurination – Sugar-base bond is spontaneously broken - Base is lost (usually purines) and nucleotide is left empty (called apurinic site) - What would happen to apurinic site during DNA replication? ______________________  Happens very often (10k a day) - See original common theme slide - Deamination - An amino group of C or A is spontaneously lost - C or A w/o amino groups wont hydrogen bond with normal G and T - DNA pol sees a deaminated C (or A) and puts in the wrong base - Same common theme
  39. 39. Your DNA is under constant assault:Science Dec. 23, 1994 Every second that you read this, the DNA in each cell of yourbody is being damaged Chemical bonds are breaking DNA strands are snapping Nucleotide bases are flying off Each cell loses more than 10,000 bases per day just fromspontaneous breakdown of DNA at body temperature Meanwhile many cells are dividing and therefore copyingDNA and each copy introduces the possibility of error Exposure to carcinogens adds to the injury and causesstrange new forms to sprout from the double helix
  40. 40. Causes of damage Spontaneous damage• Causes of spontaneous damage include: 4) Oxidative damage - Normal process of aerobic cellular respiration creates extremely reactive atoms called a free radicals - Free radicals – An atom or group of atoms that has an unpaired electron H OO H HO + OH - Free radicals will steal an electron from wherever it can get it - Proteins, lipids, DNA - Removal of electrons from DNA bases  alters their structure - If happens right before DNA replication  Common theme again!! - Thought to be major mutagen in our cells  Cancer and aging!! - Some agents can lead to increased free radical production (induced)
  41. 41. Causes of damage Spontaneous damage• Causes of spontaneous damage include: 5) Transposons (aka jumping genes) - Mobile pieces of DNA abundantly found in all living things - Nearly 45% of human genome - Cut or copy themselves and then insert randomly in the host genome - Replicative vs nonreplicative transposons - They encode enzyme transposase - Insertion near genes or within genes can disrupt host gene expression - Can also lead to larger chromosomal alterations - DNA is being cut/pasted – can go wrong - Control transposase  Control movement - Methylation and mRNA destruction http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#
  42. 42. Causes of damage Induced damage• Some external agents (chemical and physical) can induce DNA damage: 1) Base analogs - Chemicals that resemble normal nucleotides and can substitute for them during DNA replication - However, they exhibit abnormal base-pairing properties - Example: 5-bromouracil resembles thymine - DNA pol will incorporate 5BU instead of thymine during DNA rep - 2nd round of rep – DNA pol puts a "G" across from 5BU - 3rd round of rep – wrong "G" serves as template for wrong "C"
  43. 43. Causes of damage Induced damage• Causes of induced damage include: 2) Alkylating agents - These chemicals add an alkyl group (CH3 or CH3CH2) to amino or ketone groups in nucleotides - Alkylated nucleotides exhibit abnormal base pairing - Ex: Ethyl guanine pairs with T - Two rounds of DNA rep  Mutation (same theme) GC GC GT + GC AT + GT - Mustard gas (structure above) – Alkylating agent used as a weapon in WWI - Soldiers came down with severe burns, blindness, and tumorsCurrent chemical attacks on unsuspecting populations: http://www.youtube.com/watch?v=bwJKYHNGT98&feature=related
  44. 44. Causes of damage Induced damage• Causes of induced damage include: 3) Intercalating agents - Flat, multiple-ringed molecules that tightly wedge themselves betweenthe bases of DNA  distorts its 3-D structure - DNA pol gets confused  adds or removes a nucleotide - Cause insertions or deletions in the DNA (unlike all others discussed) - Examples include acridine orange and ethidium bromide - They are common used to visualize DNA during centrifugation or gel electrophoresis
  45. 45. Causes of damage Induced damage• Causes of induced damage include: 4) UV light and low energy radiation - All electromagnetic radiation having wavelengths shorter than visible light (~380 nm) are very energetic - Disrupt DNA and other macromolecules - UV light  λ≈260 nm and is very mutagenic - UV light causes adjacent pyrimidine bases to fuse with one another - Called pyrimidine dimers (usually two thymines) - Distort DNA 3-D structure - Pyrimidine dimers prevent DNA pol from replicating normally - Insert wrong, too many, too few - Cells containing too many of these dimers will kill themselves via cell suicide (apoptosis)
  46. 46. Causes of damage Induced damage• Causes of induced damage include: 5) High-energy radiation (ionizing radiation) - Electromagnetic radiation with shorter wavelengthseven worse: - X-rays, gamma rays, cosmic rays - Mutates DNA in different ways: 1) It cause electrons to be released from various molecules in the cell producing free radicals - This is called ionization - Free radicals mutate DNA as described 2) It directly breaks phosphodiester bonds in the DNA strands (causes double- stranded breaks) - Can produce deletions, translocations, inversions 3) Creates thymine dimers - Why do we treat tumors with X-rays?
  47. 47. Causes of damage Induced damage• Causes of induced damage include: 6) Viruses - Retroviruses have the ability to randomly insert themselves into our genome - Usually go into junk (no issue) - If go into a promoter or coding sequence  gene expression disrupted - Similar to transposons (gigantic insertion) - EXAMPLE: Retroviral gene therapy and leukemia - Other viruses produce proteins that directly inhibit DNA replication, monitoring, or repair mechanisms - Indirectly encourages mutations to be introduced - Once they are in, they cant be removed (like transposons) - Not really "damage" (damage can be fixed)
  48. 48. Assessing the mutagenicity of compounds Ames test• Ames test - Used to test if a new chemical has ability to mutate DNA (cause cancer) - Successfully identified carcinogens in hair dye (1975) - Set-up - Uses bacterial strain that cant make its own histidine (wont grow w/o it) - Mix bacteria w/ either chemical or H2O and add to Petri dish lacking histidine + H2O + chemical - No bacteria should grow to be tested + LIVER ENZYMES - Mutations can occur to allow the bacteria to make histidine  regain ability to grow - Results - H2O control  Very few colonies (spontan) - Mutagenic chemical  lots of colonies (BAD!!)
  49. 49. Repairing DNA damage• Most types of DNA damage can be fixed by the cell - Must be fixed PRIOR TO DNA REPLICATION - Remember the common theme - Exceptions: Transposons and retroviruses (cant be removed)• Different types of damage exists - Altered individual bases (alkylated, base analogs, etc...) - Altered 3-D DNA structure (thymine dimers, intercalating agents) - Double-strand DNA breaks  Different repair mechanisms must exist to detect and fix• DNA repair themes - How is it detected? - How is lesion removed/repaired? - Is any DNA cut out in the process? If so, how much? - Bacterial vs eukaryotic
  50. 50. Repairing DNA damage Direct repair• Direct DNA repair – Reverses the alteration w/o cutting out or replacing any nt - Used primarily for thymine dimers and alkylated bases• Direct repair of thymine dimers - Both bacteria and eukaryotic cells use light-dependent pathways - Eukaryotic cells – use an enzyme called photolyase to cut abnormal covalent bonds between the two thymines - Bacteria – use an enzyme called photoreactivation enzyme (PRE) to do same - PRE is activated by blue light• Direct repair of alkylated bases - Methylguanine DNA methyltransferase enzymes directly cuts off extra CH3 from guanine
  51. 51. Repairing DNA damage Indirect repair - excision repair• Excision repair – Removal of altered base/nucleotide and replacement with good DNA 1. Recognition of the lesion by 1 or more proteins and the subsequent excision of that error by a nuclease enzyme - Sometimes extra "good" sequence also removed 2. A DNA polymerase fills in the space with proper nucleotides - What enzyme would you predict does this in prokaryotic cells? 3. DNA ligase seals the final nick (the last phosphodiester bond) between the new and existing strands• Cells have 2 types of excision repair systems - Base excision repair - Nucleotide excision repair
  52. 52. Repairing DNA damage Indirect repair – base excision repair• Base excision repair - used for correction of minor alterations to individual bases (free radical, alkylated, base analog)• Mechanism (described in E.coli, but all cells have it) 1) DNA glycosylase enzymes recognize altered bases - Different glycosylases recognize different types of altered bases 2) Glycosylase then cuts out the base only (breaking the sugar/base bond) 3) AP endonuclease enzyme recognizes the nucleotide missing the base and makes a cut in the sugar/ phosphate backbone at that site 4) DNA pol I/ligase finish the job (and repair the damage)  Eukaryotic glycosylases have yet to be identified
  53. 53. Repairing DNA damage Indirect repair - NER• Nucleotide excision repair fixes larger lesions that distort the actual DNA structure and block replication - Examples: Intercalated agents, thymine dimers• NER (E.coli NER is described below) 1. DNA is damaged and a lesion forms 2. Proteins called Uvr (UvrA, B, C, D) recognize the lesion and cut it out - A-B complex recognizes the lesion - A comes off and is replaced with C - B-C together cut the DNA on either side of the lesion - Cut out extra "good" DNA on both sides - D is a helicase that liberates the cut piece 3. DNA pol I fills in the gap/ ligase seals http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#
  54. 54. Repairing DNA damage Nucleotide excision repair - Disorders• Several human disorders exist in which the NER system is defective - The best characterized of these disorders is called xeroderma pigmentosum• Xeroderma pigmentosum (XP) - Contain one of several rare mutations in some part of the NER pathway - They have severe skin abnormalities when exposed to the sun - UV light exposure Induces freckling, ulceration, and skin cancer - Why is the sun so damaging? - Produce 1000s of TT a day - No repair  mutation  cells die or become cancerous - Scientists isolated DNA from XP patients and attempted to find which gene was mutated - Found that mutations in any of 7 different genes all lead to XP  NER in eukaryotic cells involves as many as 20 different proteins
  55. 55. Repairing DNA damage Indirect repair - mismatch repair• Mismatch repair  fixes mismatches (DNA may G look okay otherwise) T• Problem: If the cell has a G-T mismatch, how does it know which one is correct? ( the G or the T) Which strand - Hint #1: Mismatches usually appear following DNA is wrong? replication - Common theme review  "Wrong" nucleotide is always on the new strand - Hint #2: Newly-made DNA strands stay unmethylated for a little while - New and old DNA strands look different for a short time (hemi-methylated) - If wait too long, both become methylated  Wrong nucleotide is always on the new, unmethylated strand!!!
  56. 56. Repairing DNA damage Indirect repair - mismatch repair• Mismatch repair - DNA commonly contains methylated adenines - No effect on transcription (cytosine CH3) - Adenine methylase add CH3 when seeing GATC - Mechanism (E. coli) 1) MutS protein locates mismatches - Forms complex with MutL afterward (linker) 2) MutL binds to MutH, which is bound to a nearby hemi-methylated site - DNA must loop out to allow L-H interaction 3) MutH makes a cut in the unmethylated strand 4) MutU acts as a helicase to release the unmethylated strand before an exonuclease destroys it 5) DNA pol III fills in with proper sequence, ligase seals http://www.youtube.com/watch?v=ESBL6Qxsi90
  57. 57. Repairing DNA damage Fixing double-stranded breaks• Most repair pathways require 1 good template strand - What is done when both strands damaged?• Two repair pathways fix double-stranded breaks: 1) Homologous recombination repair (E.coli) a) Homologous chromosome first brought in - Usually the sister chromatid b) RecBCD recognizes double stranded breaks - Partially degrades 1 strand on each side - Creates single-stranded overhangs c) RecA binds to single-stranded end and promotes invasion of the homologous chr. - The good strand loops up (D-loop) d) RuvABC, DNA polymerase, and ligase help to recreate the gaps and resolve the structure  The once damaged chromosome will contain a piece of the homologous chr.  Very similar to what happens during crossing over
  58. 58. Repairing DNA damage Fixing double-stranded breaks• Two repair pathways fix double-stranded breaks: 2) Non-homologous end-joining - The two broken ends are simply glued back together - No requirement of sister chromatid - End-binding proteins bind to each side of the break (to stabilize) - Cross-bridging proteins recruited to prevent drifting of the two pieces - Ends are processed, filled, and ligated - Advantage  Can happen any time in cell cycle (no sister chr. required) - Disadvantage  Can lead to small deletions near the break site (result of processing)
  59. 59. Repairing DNA damage Translesion synthesis• Some lesions (e.g. TT) block normal DNA replication (via DNA pol III) - If other repair fails, translesion synthesis will initiate to allow DNA replication to finish• Translesion synthesis (called SOS repair in E. coli) - Stalling of normal DNA polymerase by lesion triggers recruitment of "emergency" polymerases - Have different binding pocket  more tolerant of altered DNA structure - Emergency pols (e.g. DNA pol II, IV, V) replicate over the lesion - Problem: They are very error prone - DNA gets replicate, but with mistakes - Original lesion remains (not fixed) - Translesion synthesis enables rep to continue
  60. 60. Benjamin A. Pierce GENETICS A Conceptual Approach FOURTH EDITION CHAPTER 19 Molecular Genetic Analysis and Biotechnology© 2012 W. H. Freeman and Company
  61. 61. Chapter 19 Outline19.1 Techniques of Molecular Genetics Have Revolutionized Biology, 51419.2 Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes, 51519.3 Molecular Techniques Can Be Used to Find Genes of Interest, 527
  62. 62. Chapter 19 Outline19.4 DNA Sequences Can Be Determined and Analyzed, 53319.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function, 54119.6 Biotechnology Harnesses the Power of Molecular Genetics, 547
  63. 63. Locating DNA Fragments with Southern Blotting and Probes• Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest
  64. 64. 19.3 Molecular Techniques Can Be Used to Find Genes of InterestGene LibrariesIn Situ Hybridization
  65. 65. Gene LibrariesDNA library: a collection of clones containing all the DNA fragments from one source • Creating a genomic DNA library • cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA
  66. 66. Gene LibrariesDNA library: a collection of clones containing all the DNA fragments from one source • Creating a genomic DNA library • cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA
  67. 67. From protein to DNA
  68. 68. •Gene libraries finding genes of interest- Paradoxically, researchers must first clone aGene in order to “find” it- What are the advantages and disadvantages of:- Creating a genomic library using partial digestionWith restriction endonucleases- Creating a cDNA library
  69. 69. • Screening Gene libraries:- Usually done using a probe from a similarGene isolated from other species and hybridizing- Can also deduce DNA sequence from knownProtein sequenceIn this case, a mixture of all possible nucleotidescombinations is used as probe.- May also look for the protein product of a gene(Western Blot: specific antibodies)
  70. 70. Genomic and cDNA libraries can be screened with a probe to find the gene of interest.
  71. 71. Site-Directed Mutagenesis• Oligonucleotide-directed mutagenesis
  72. 72. Transgenic Animals• Transgene
  73. 73. Knockout Mice• A normal gene of the mouse• has been fully disabled.• Knock-in mice: a mouse carries• an inserted DNA sequence• at specific locations