BMB 462 Lecture 25 Outline of Last Lecture I Continuing Elongation a Review b Lagging strand Primer removal and Nick sealing II Termination III Chromosomal Separation IV Eukaryotic DNA Replication a Complications Replicating Eukaryotic Chromosome Ends V The Central Dogma VI Polymerase Inhibitors Outline of Current Lecture I II III IV V Mutations and Carcinogens DNA Repair Mechanisms Overview Mismatch Repair Base Excision Repair Nucleotide Excision Repair Current Lecture Concepts to remembers from previous courses lectures A mutation is a permanent change in the sequence of DNA I Mutations and Carcinogens a In the genetic code you have a base triplet a codon that encodes the amino acid The code is redundant meaning that more than one codon can code for the same amino acid b Types of Mutations i Silent the mutation is either synonymous aka it codes for the same amino acid or it is silent because it doesn t change the phenotype i e if the mutation is not in the exons ii Missense the mutation causes the codon to signal for a different amino acid These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute iii Nonsense the mutation turns the codon into a termination signal which ends translation iv Frame shift base s are added or deleted shifting the codon sequences This can either result in a nonsense or missense mutation c The Ames Test testing Carcinogens i This test uses a bacterial salmonella strain that cannot grow in the absence of Histidine 1 Plate A is media without histidine You do have some colonies growing due to normal mutation rate but there are not many 2 Plates B D The Ames test adds a mutagen disk to see if the mutagen causes mutation It is typically used to test carcinogens since carcinogens increase the mutation rate They cause mutations so that more colonies can grow in the absence of histidine ii Kill Zone the area around a mutagen where it is too concentrated for the strain to grow d There is always mutation sometimes too much mutation will significantly impact the function of the cell and it will die II DNA Repair Mechanisms Overview a Using the complementary strand i I e mismatch repair this is typically the first repair strategy used repairs are made almost immediately 1 Repair mismatches as they re incorporated into the newly synthesized strand ii Base excision removing the incorrect base from the backbone 1 Repair abnormal bases i e deaminated bases alkylated oxidized iii Nucleotide removing the entire nucleotide b Direct Repair i This mechanism doesn t remove the mistake instead they fix the error in place 1 i e they reform the base into the correct conformation c Using a Homologous Chromosome i Use the 2nd double helix to repair the first ii The bacteria use the replicated chromosome to repair mistakes 1 i e recombination repair d Having something is better than nothing i Nonhomologous end joining you have a break in the chromosome and join the 2 ends together It repairs the double stranded DNA breaks 1 This method can be accurate but is not the most efficient manner ii Error prone translesion DNA synthesis they read over the mistake and continue on with replication since something is better than nothing iii Both of these would involve the SOS repair mechanism when nothing else could be done to fix the error III Mismatch Repair a Polymerase III makes 1 error in 104 bases Proofreading decreases that to 1 in 106 108 i Immediately after replication 1 in 109 1010 b E coli repair is methyl directed After synthesis the DNA is hemi methylated Before the cell methylates the newly synthesized strand it has the opportunity to go in and find any mismatches The cell knows that the methylated strand is the correct one c Beginning the repair process MutS and MutL find the mismatch d Finding hemimethylated sites MutH binds to the complex and scans the DNA e Cleaving the damaged strand MutH binds to the new strand the nonmethylated strand and cleaves it i Where it gets cleaved depends on where MutH first finds the GATC sequence to the right or left of the mismatch f Removing and replacing DNA For removing or replacing The process is different depending on where the mismatch is relative to the MutH bound on GATC i Depends on where it encounters that methylated GATC first ii Different enzymes are involved depending on where the nick is relative to the mismatch 1 MutH is an endonuclease that makes a nick in dsDNA cleaves only one strand 2 Other endonucleases called restriction enzymes make a nick in both strands of the dsDNA iii Step 1 Requires an endonuclease i e MutH 1 It can cut a single strand of DNA in a double stranded complex It only cuts one of the strands but the strands can be bonded together 2 Restriction enzymes make cuts in both strands of the double helix a The nick is 3 of the mismatch so it requires a 3 5 exonucleases b If the nick is on the other end 5 of mismatch you need a 5 to 3 exonuclease iv This isn t the most efficient repair mechanism since the closest methylated GATC sequence can be thousands of base pairs away It is a very costly means of repairing a single mismatch 1 This emphasizes the importance of conserving DNA sequence and fixing mismatches before they become mutations v Some mutations are more prone to causing cancer 1 In humans damage to MutL or MutS predisposes one to cancer HNPCCC hereditary non polyposis colorectal cancer IV Base Excision Repair a Removes a certain type of damaged base i i e deaminated bases spontaneous chemical reactions can deaminate cytosine to uracil When a uracil is found in the DNA sequence the cell knows it should likely be repaired by replacing it with cytosine 1 Deaminating adenine creates hypoxanthine 2 Deaminating guanine xanthine 3 Deaminating thymine thymine can t be deaminated because it doesn t have an amine group Deaminating 5 methylcytosine will create thymine though 4 Mononucleotides i e IMP and XMP hypo xanthine are intermediates in purine nucleotide metabolism ii If uracil is not repaired it will most likely be recognized as a thymine and will be paired with adenine This causes a GC base pair to be mutated into an AT base pair b Recognition and removal of damaged bases First the base is cleaved removing the damaged deaminated base i The base is removed from the backbone creating a debased site 1 A family of DNA glycosylases does this each recognizes specific damage a Cleavage of glycosidic bond results in
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