BSCI222 – Lecture 22 (11/21/13)- A molecular characterization of mutations- Base substitution versus insertion or deletion: substitution = one letter changed for another; insertion and deletion inserts new ones or deletes and changes the line-up (can be any number of nucleotides.o Triplet repeats are one of the most common sources of human disease. There is a normal range of number of copies (muscular atrophy: 11-33 copies is normal, 40-62 copies is in the disease range, protein nonfunctional.)o Once a certain number of triplets is reached, tends to keep going and expanding. Happen because of a mis-pairing during replication (the hairpin loop forms, and the legs are matched correctly on the strand, but the rest of the loop is mismatched, and every time that strand serves as a template now, each molecule will have more repeats.- Transitions: A G or C T; transversions are going from purine to pyrimidine or pyrimidine to purine (A C, T A, T G, etc.)- Mutations characterized by phenotype:o Missense mutation: changes the amino acid being encodedo Nonsense mutation: introduces a stop codon in the middle of the sequenceo Silent mutation: mutation in the third base of a codon, no change in amino acid sequence. o Forward mutation: changes the wild-type into a mutant phenotypeo Backward/reverse mutation: restores the wild-type gene and phenotypeo Suppressor mutation: can happen after a forward mutation, suppresses the mutant phenotype, get wild-type phenotype. Individual has both the original and the suppressor mutation, with a wild-type phenotype. Intragenic suppressor mutations: second mutation in the same gene to suppress the mutation. Intergenic suppressor mutations: base substitution  stop codon  wouldnormally stop the process, get a short nonfunctional protein. But in yeast and E. coli and humans, can often reverse the effects of that (suppressor) through a mutation in the tRNA gene (if you have a mutation that allows itnow to recognize the nonsense mutation, and can translate the protein again). Suppressor tRNA.- Don’t want tRNA to do this all the time, really only happens in a small genetic system where you aren’t extending that many proteins.o Frameshift mutation: insertion or deletion that alters the reading frame of a gene.- Radiation high-energy particles break the DNA strands. But, most mutations are arising in the normal biochemistry of the cell.o Errors in replication: Sometimes when the polymerase goes by, the DNA is in its rare enol form instead of the common keto (Watson and Crick’s assumptions). When the Polymerase goes by, because of the different H-bonding, might put in Ato pair with a C, and a T to pair with a G. Turns out to not be a very common mechanism of mutation though Can also have wobble base mis-pairing. A wobble mispair  one strand replicates normally, the other strand introduces the wrong base pair. After another round of replication  new sequence in that region (takes 2 rounds of replication for a fully mutant helix, only on one strand after one round of replication).  Insertion/deletion of slippage: newly synthesizes strand loops out, resulting in the addition of one nucleotide on the new strand. Template strand loops out  mutation.o Spontaneous damage to bases:  Spontaneous depurination: the base gets cleaved off the sugar phosphate backbone, leaving the backbone intact but the base is gone (called an abasic site or apyrinic or apyramidic, AP) If the AP site isn’t repaired, this leads to mutation. Most polymerases, when presented with this problem, insert an A.  Deamination: Cystosine minus NH2  Uracil (enzymes can recognize it, fix it). 5-methylcytosine minus NH2  Thymine (no way the enzymes in the cell can recognize this as a mutant base, looks completely normal to them. Particularly difficult to repair because isn’t being flagged as broken.) Get about 100,000 base pair lesions per cell per day. As you age, repair systems can’t keep up, starts to affect gene expression. o Chemically-induced damage to bases: 5-Bromouracil (base analog, looks a lot like a Thymine, has a Bromine atom on it that has an abnormal pairing, so while the normal pairing wouldbe mimicking an AT base pair, the way the Bromouracil is usually found in an ionized form, will end up pairing with G.) This is a base substitution,specifically (A G, purine  purine) transition. Lots of different kinds of chemical damage can cause substitutions. Antioxidants are good because they soak up reactive Oxygen species (reacts with your DNA). Hydroxide free radicals attack a DNA base and change its structure, insert into bases. Changes the H-bonds, changes how it pairs. Modify Guanine by letting a hydroxide radical, make an 8-oxy-7-8-dihydrodeoxyguanine (may mis-pair with adenine). Intercalating agents: mimic a base pair, distort the helix shape, wind up making insertion/deletion sites. UV light: changes the bonds in adjacent Thymines (two next to each other in a sequence  UV crosses them together, creating covalent bonds, Thymine dimer. If they’re paired to each other, obviously won’t pair to theA’s on the opposite strand. Can’t replicate or will replicate incorrectly, because helix is distorted and the Thymines aren’t available.o Repair systems:o Mismatch repair: If a polymerase comes across a pair of bases that are mismatched, how does a repair system know how to fix that? Which one is the correct/original one? Hard to tell. The way your cell figures it out is that it goes and looks for one of these sites where a methyl group has been added (after replication, the DNA gets methylated at those sites, GATC sites). Part of the mismatch repair complex first binds to the methylated sequence (only the old strand is methylated immediately after replication, so repair system is bound to only a single strand). The damaged site gets brought into the multiprotein repair complex, which is keeping track of which strand is the original, and makes the repair on the new strand (cleaves out that region, chews it away, and does the synthesis again). Can only be done in the short period of time after replication, while old strand is still methylated and new strand has not yet been methylated.o Direct repair: Fixes pyrimidine dimers, “suicide enzymes”. An alkalizing agent has added an extra methyl group to a base  would like to remove that; suicide enzymes go in and react with the methyl group and