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Mutation and DNA Repair Mutation Rates Vary Depending on Functional Constraints Low Mutation Rates are Necessary for the Evolution of Complexity 1. Because most mutations are deleterious, there are limits to the number of mutations that an organism can afford to accumulate in its somatic body, e.g., a) given mean eukaryotic rates, genomes can accommodate 60,000 genes without intolerable mutational loads (Alberts et al.) b) a mutation rate 10 times higher would limit genome size to ca. 6000 genes 2. Both the germ line and the somatic body must be protected from mutational load (rare mutations become common because of large genomes and cell proliferation), e.g., a) germ line: (1) (2) (3) (4) DNA repair meiotic recombination in all eukaryotes sequestering of germ line in metazoans diplontic selection among cell lineages in meristems of plants b) somatic tissues ...20% of deaths in western societies are due to cancer (uncontrolled cell proliferation) resulting largely to the accumulation of genetic damage in somatic tissues (1) DNA repair (2) immune systems 3. less efficient DNA repair and absence of meiosis may explain the limitation of prokaryotes to small genomes and unicellular forms before the origin of these processes in the protoeukaruote line. 4. spontaneous nucleotide changes are much higher than mutation rates would indicate, because of DNA repair mechanisms Two strategies to study gene function • Genotype to Phenotype - sequencing and searching for homologous sequences, then study their function • Phenotype to Genotype - mutational screens and functional analysis Kinds of Mutations • Point Mutations – Same sense mutations – Missense Mutations – Nonsense Mutations – Transitions – Transversions • Frame shift mutations • Substitutions, Deletions and Additions Chemistry of single nucleotide substitutions: a) transitions: a pyrimidine replaces a pyrimidine (C T or T C) or a purine replaces a purine (A G or G A) b) transversions: a pyrimidine replaces a purine or vice versa c) transitions are less severe mutations that transversions: (1) chemically, purines are more similar to one another than they are to pyrmidines, and vice versa (2) genetically, amino acid substitution is less likely with transitions because of the degeneracy of the genetic code (a) 3rd position transitions often code same amino acid i) UUU and UUG both code for leucine ii) GAA and GAG both code for glutamic acid (b) 3rd position transversion less often codes for same amino acid i) UUU and UUG code for phenylanaline and leucine Mutagenesis • Spontaneous Mutations – Replication Errors – Other Errors • Chemical Mutagenesis • Radiation-induced Mutations Replication Errors Replication Proofreading Mutator Strains of E. coli • error prone replication • mutD codes for e subunit of DNA pol III: DNA polymerase III holoenzyme with subunits (weight in daltons) e Step 1: previous nucleotide pair is tested for complementarity. If passed, elongation occurs. Step 2: If failed, the elongating strand is transferred to the exonuclease site to excise the mismatched nucleotide. Experimental Demonstration of Proofreading double labeled probe artificial template non-complementary nucleotide excised, but no complementary nucleotides last nucleotide is noncomplementary and labeled Tautomerization of Bases Thymine Tautomers: T•A to T•G binding mutation from T to A Replication A • replication Tketo A • replication Tenol A template • Tketo daughter A daughter • Tketo template A template • Tketo daughter G daughter • Tenol template replication AT replication AT replication AT replication AT replication AT replication AT if unrepaired replication replication GC AT Adenine Tautomers : A•T to A•C binding mutation from C to T Replication Aamino • replication T Aimino • replication T Aamino template • T daughter Aamino daughter • T template Aimino template • C daughter Aamino daughter • T template replication AT replication AT replication AT replication AT replication AT if unrepaired replication GC replication AT replication AT Cytosine Tautomers : Camino•G Cimino•A binding mutation from C to T common results in C•G pairing rare results in C•A pairing AT substitution Replication Camino • replication G Cimino • replication G Camino template • G daughter Camino daughter • G template Cimino template • A daughter Camino daughter • G template replication CG replication CG replication CG replication CG replication CG if unrepaired replication TA replication CG replication CG Guanine Tautomers : Gketo•C Genol•T binding mutation from G to A common results in G•C pairing rare results in G•T pairing Replication Gketo • replication C Genol • replication C Gketo template • C daughter Gketo daughter • C template Genol template • T daughter Gketo daughter • C template replication GC replication GC replication GC replication GC replication GC if unrepaired replication TA replication GC replication GC Frameshift Mutations insertion Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication Spontaneous Mechanisms Outside of Replication Spontaneous hydrolysis can result in deamination and depurination Deamination replacement of an amino group by a carbonyl oxygen These nucleotide analogs have different pairing affinities, but analogs can be recognized and repaired 5-methyl C deamination results in T, which can’t be recognized as a mutation Replication produces a GC and an AT C’s are selected for methylation in certain CG sequences, which has led to the conversion of most CG’s to TG’s during evolution Deamination of C and A illustrating different pairing behavior Deamination and repair of C Deamination Repair of a Deaminated Cytosine Deamination of 5-methylcytosine Triplet Repeats Pathology results when repeats exceed a threshold number. Amplification of copy number by unequal crossing-over Unequal crossing-over becomes more likely with increased copy number Dynamic Mutations Unequal crossing-over becomes more likely with increased copy number and The severity of the pathology increases with copy number therefore... Both the probability of the pathology and its severity increase over generations after the number of repeats approaches the threshold A number of conditions are based on this mechanism operating in different genes The repeats can be located in different orientations with regard to the coding sequence upstream downstream within within The repeats can be located in different orientations with regard to the coding sequence ...even within a single gene Chemical Mutagenesis EMS is an alkylating agent Nucleoside analogs can exhibit variant pairing behavior keto (above); enol pairs to G instead of A Acridine dyes intercalate DNA sequences Effect: stabilizes the looping that leads to deletions and insertions that cause frame shift mutations Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication Major Repair Mechanisms • Mismatch repair • Excision repair • Double strand breaks repaired mainly by end-joining • Inducible & error-prone mechanisms Excision Repair Excision repair mechanism More excision repair modalities Repair of UV damage Thymine dimers Excision Repair of UV Induced Thymine Dimers Mismatch Repair Mismatch Repair • To catch single-base errors that slip through proofreading during replication • Happens right after replication • Misses C•C and small insertions and deletions • mutH, mutL, mutS mutator strains are involved in mismatch repair • Trick is distinguishing the new daughter strand MutH Missmatch Repair How is the daughter strand recognized as the strand to correct? GATC sequences methylated on the 6 position of the A base Mismatch Repair Mismatch Repair endonuclease Activity …nicks DNA and then methylation Radiation Induced Mutagenesis • UV induced Thymine dimers • Gamma and X-ray double stranded breaks Spectrum X-rays induce mutations Multiple mechanisms to repair UV damage Photo-activated Repair System Repairing Doublestranded Breaks • often caused by radiation (high energy gamma or X-rays, directly or by creation of free radicals) • repaired by: ◊ Homologous recombination ◊ Blunt-end repair (right) problem: deletion of short nucleotide sequence Inducible Repair • Backup systems activated only in emergencies • Inducible • Error prone SOS Undoing alkylation Note that the enzyme is expended! A tangible example of the importance of DNA repair Photo-activated Repair System Recombination Repair bulky mutations can leave gapsafter replication p53 activation of DNA repair