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 encoded o Nonsense mutation introduces a stop codon in the middle of the sequence o 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 phenotype o Backward reverse mutation restores the wild type gene and phenotype o 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 would normally 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 it now 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 A to 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 o Chemically induced 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 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 would be 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 the A 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 remove it have a cystine reacts with the methyl group Called a suicide enzyme because it s not really an enzyme after the process is done it s stuck with the methyl group forever can never repair anything ever again has to be chewed up Very expensive process because you re making an entire protein