BIOLOGY 305 1st Edition Lecture 20 Outline of Last Lecture I. Introduction to TranslationII. Mechanism of DNA TranslationA. InitiationB. ElongationC. TerminationIII. Genetic CodeA. 3 Features of the Genetic Code & 2 ExperimentsB. Code Bias and The Wobble Effect IV. Vocabulary and Sample QuestionsOutline of Current Lecture I. MutationsA. ProofreadingB. Post-Replication RepairC. Spontaneous MutationsD. Induced MutationsII. Seven Forms of Repair: OverviewA. Mismatch Repair, Direct ReversalB. Base Excision Repair, Nucleotide Excision RepairC. Translesion Synthesis, NHEJ, RecombinationIII. Types of Mutations Affecting Coding and their EffectsIV. DiseasesV. Vocabulary and Sample QuestionsCurrent LectureRecall: In translation, 5’-3’ terminals of mRNA = N-C terminals of protein formedI. MutationsKey traits of genetic material: Ability to contain information, ability to replicate, ability to mutate, regulationOrder of Error Probability in DNA replication mechanisms:3’ to 5’ proofreading < mismatch repair < 5’ to 3’ polymerase activity and base selection/pairingOverall fidelity is still only 1 error per 1010 nucleotidesA. Proofreading: proofreading is associated with replicative DNA polymeraseThe 3’ to 5’ activity of exonuclease (of DNA ) polymerase rapidly removes incorrect base: This diagram shows the directionality of the polymerase as it moves from 5’  3’ while proofreading from 3’  5’. The 3’ to 5’ activity of exonuclease activity rapidly removes incorrect base, and DNA synthesis resumesB. Post-replication repair: repairs errors missed by the proofreading stage in replicationEx: Methyl directed mismatch repair in E. ColiThe template strand is first methylated for protection while the a piece of the new strand is cut out, then DNA polymerase and ligase reassociate to fix the daughter strand, which then becomes methylated as wellDifferent Mut proteins have different recognitions and jobs:MutS  mismatch sensorMutL  linkerMutH  Endonuclease nicks the new strandIf mismatch repairs are left undone, they have elevated frequencies: for example, isolation of two single colony mutants of E Coli resistance to a plate with drugs transferred to a new plate with same drug shows an overwhelming growth of mutant coloniesC. Spontaneous Mutations:There are three types of spontaneous mutations:1) Depurination – the purine or pyrimidine is removed from the DNA backbone (shown: removal of guanine)2) Deamination – an amido group is altered or oxidized, such as conversion of C to U (wrong base is formed as a result)3) Oxidative damage (wrong base pairing occurs)D. Induced Mutations:Base Analogs (such as hydroxylamine) vs. Base mutations – both result in correcting base pairingIntercalating agents – molecules like proflavin or EtBr that fit in between the bases and alter its structure (results in insertions and deletions)Base damage – incorrect formation of bases (such as thymine dimers caused by UV rays) resultsin wrong structuresBackbone breaks – breaks in the phosphodiester backbone of DNAHydroxymethylcytosine – a DNA pyrimidine nitrogen base formed on cytosine that is important to epigenetics that has a potential ability to turn genes on and off:II. Seven Forms of Repair Overview1) Mismatch Repair – a system for recognizing and repairing erroneous insertions, deletions, and other base errors in DNA replication, recombination, and repair. The system is strand specific and able to recognize the old strand from the new, uses DNA methylation on strands of DNA to recognize errors2) Direct Reversal – recognizes changes in base structure, does not require a template because the types of damage that it is able to counteract can only be one of the four bases, does not involve breakage of the phosphodiester backbone, 400nm light activates reparative photolyase3) Base Excision Repair – removes small, non-helix distorting base legions from genome, recognizes changes in base structure, initiated by DNA glycosylates 4) Nucleotide Excision Repair – recognizes change in base structure, repairs bulky, helix distorting base legions, both this type and base excision repair involve proteins sliding along theDNA helix and attaching to damaged areas5) Translesion Synthesis – a DNA damage tolerance process that allows replication past lesions such as AP sites and thymine dimers, DNA polymerase is briefly switched out for a specialized translesion polymerase (Bypass Polymerase), does not have features that recognize errors and isa last-resort method, also has a high mutation rate6) Non-Homologous End Joining (NHEJ) – called non-homologous because break ends are ligated without a need for a homologous template, single-stranded overhangs guide repair and often determine compatibility7) Recombination – recognizes errors in the sister chromatid: the ends of the erroneous chromatid are trimmed, the sister stand ‘invades’ and forms a D-loop which activates DNA synthesis to occur, then the new DNA unwinds and anneals to form correctly before ligatedIII. Types of Mutations Affecting Genetic Coding and EffectSilent mutations – result in unchanged gene products (mostly due to wobble in 3rd codon)Missense – a change in base usually in the 1st or 2nd base of the codon, resulting in changed amino acidsNonsense – mutation results in a STOP codon in the mRNA, which results in polypeptides ending prematurelyFrameshifts - single insertion/deletion that vary, the closer toward the 5’ end of of the mRNA, the more devastating the effect is likely to be on the polypeptideRegulatory mechanismsMutations in…Promoter  change in gene expressionRegulatory regions  change or blocking of formation of gene productsSplicing acceptor, splice donor, branch sites  disruption of splicing5’-UTR or 3’ UTR  translation of mRNA is affectedSummary (Know what each mutation looks like on a Northern vs. Western Blot!!)Missense – pyrimidines  purines = amount of RNA and protein unchangedNonsense – premature STOP codon = RNA unchanged, but protein is are shorter/fartherFrameshift - deletion/insertion of one base pair = mRNA should be unaffected but the protein can vary depending on the location of the mutation or sequence affectedRegulatory-region mutations – no mRNA results thus no protein results, nothing can be seen ongelIV. DiseasesCancer:Clonal – in all tumor cellsSubclonal – in a subset of tumor cells (most mutations are