BMB 462 Lecture 25 Outline of Last Lecture I. Continuing Elongationa. Reviewb. Lagging strand Primer removal and Nick sealingII. TerminationIII. Chromosomal SeparationIV. Eukaryotic DNA Replicationa. Complications Replicating Eukaryotic Chromosome EndsV. The Central DogmaVI. Polymerase InhibitorsOutline of Current Lecture I. Mutations and CarcinogensII. DNA Repair Mechanisms – OverviewIII. Mismatch RepairIV. Base-Excision RepairV. Nucleotide-Excision RepairCurrent LectureConcepts to remembers from previous courses/lectures:- A mutation is a permanent change in the sequence of DNAI. Mutations and Carcinogensa. 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 thesame amino acid.b. Types of Mutationsi. 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 – Overviewa. Using the complementary strandi. I.e. mismatch repair – this is typically the first repair strategy used; repairsare 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, oxidizediii. Nucleotide - removing the entire nucleotideb. Direct Repairi. 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 Chromosomei. Use the 2nd double helix to repair the first. ii. The bacteria use the replicated chromosome to repair mistakes.1. i.e. recombination repaird. Having something is better than nothingi. 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 Repaira. Polymerase III makes 1 error in 104 bases. Proofreading decreases that to 1 in 106-108i. 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 togo 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 mismatchd. Finding hemimethylated sites - MutH binds to the complex and scans the DNAe. Cleaving the damaged strand - MutH binds to the new strand (the non-methylated 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 differentdepending on where the mismatch is relative to the MutH bound on GATC.i. Depends on where it encounters that methylated GATC firstii. 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 onlyone 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 cancerIV. Base-Excision Repaira. Removes a certain type of damaged basei. 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 cytosine1. Deaminating adenine creates hypoxanthine2. Deaminating guanine - xanthine3. 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 intoan AT base pair.b. Recognition and removal of damaged bases - First the base is cleaved, removing the damaged, deaminated
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