1. Mutations, Mutagenesis, and Repair Chapter 10

2. The Problem DNA extremely long, fragile DNA extremely long, fragile Subject to both physical and chemical damage Subject to both physical and chemical damage Consequences could be lethal for organism or offspring Consequences could be lethal for organism or offspring

3. Mutation A heritable change in the base sequence of DNA A heritable change in the base sequence of DNA Point mutation- change in a single base position Point mutation- change in a single base position Additions Additions Deletions Deletions Substitutions Substitutions Transitions Transitions Transversions Transversions Multiple mutations Multiple mutations Frameshift mutations }

4. Consequences of Mutation Silent Mutation---base change, no amino acid change Silent Mutation---base change, no amino acid change Neutral Mutation--- Base change resulting in aa change that does not affect protein function Neutral Mutation--- Base change resulting in aa change that does not affect protein function EX. Apartic acid (D)  Glutamic acid (E) EX. Apartic acid (D)  Glutamic acid (E) Missense mutation---altered codon, new aa with different chemical properties. Function affected. Missense mutation---altered codon, new aa with different chemical properties. Function affected. Nonsense mutation---base pair substitution results in a stop codon (and shorter polypeptide) Nonsense mutation---base pair substitution results in a stop codon (and shorter polypeptide) Frameshift mutations—additions or deletions. Peptide may be longer or shorter. Frameshift mutations—additions or deletions. Peptide may be longer or shorter. Sense mutation? Sense mutation?

5. Other Terms Conditional Mutation—wild type function except under certain (permissive) conditions Conditional Mutation—wild type function except under certain (permissive) conditions Ex. Temperature sensitive mutants show mutant phenotype only at certain temperatures Ex. Temperature sensitive mutants show mutant phenotype only at certain temperatures Leaky mutations— a missense amino acid change that reduces but doesn’t eliminate protein function Leaky mutations— a missense amino acid change that reduces but doesn’t eliminate protein function

6. Mutagenesis The process of mutation The process of mutation Mutagen—anything that promotes ort causes mutations Mutagen—anything that promotes ort causes mutations Chemical Chemical Physical Physical

7. Mutation-Causes Incorrect base pairing due to tautomeric shifts Incorrect base pairing due to tautomeric shifts Removal of nitrogenous bases Removal of nitrogenous bases Alteration of nitrogenous bases Alteration of nitrogenous bases Addition or deletion of nucleotides Addition or deletion of nucleotides Single strand breaks Single strand breaks Double strand breaks Double strand breaks Crosslinking—covalent linkage between bases Crosslinking—covalent linkage between bases

8. Spontaneous Mutations Arise without mutagenic agents. DNA pol has proofreading function, can remove mismatched base Arise without mutagenic agents. DNA pol has proofreading function, can remove mismatched base Even if DNA pol misses a mismatch other systems can recognize and repair it. Even if DNA pol misses a mismatch other systems can recognize and repair it. Recognition? Recognition? Hemimethylation-allows enzymes to distinguish between parent and daughter strands. Hemimethylation-allows enzymes to distinguish between parent and daughter strands.

9. Spontaneous Mutations Tautomeric shifts during replication. Tautomeric shifts during replication.. Depurination—if a purine base is lost from C-1 of deoxyribose, will get apurinic site. Depurination—if a purine base is lost from C-1 of deoxyribose, will get apurinic site.  Odds of misincorporation on the daughter strand=75%  Odds of misincorporation on the daughter strand=75% Enzymes specific for this type of mutation have evolved Enzymes specific for this type of mutation have evolved Deamination. Deamination. C  U C  U A  Hypoxanthine A  Hypoxanthine Altered H-bonding }

10. Tautomers and Mutation Rare imino forms of adenine and cytosine Rare enol forms of thymine and guanine Normal base pairing Back

11. Deamination of C and A C  U C  U 3 H- bonds w/G  2 H-bonds w/A 3 H- bonds w/G  2 H-bonds w/A A  Hypo- xanthine A  Hypo- xanthine 2 H- bonds w/G  3 H-bonds w/C 2 H- bonds w/G  3 H-bonds w/C

12. Removing and Replacing Uracil Uracil automatically removed from DNA by uracil N-glycosylase Uracil automatically removed from DNA by uracil N-glycosylase AP Endonuclease cuts 5’ to apurinic site AP Endonuclease cuts 5’ to apurinic site Sugar phosphate removed by phosphodiesterase Sugar phosphate removed by phosphodiesterase DNA pol I adds correct base DNA pol I adds correct base Ligase seals Ligase seals Base Excision Repair (BER) Base Excision Repair (BER)

13. Base Excision Repair (BER) P A P G P G P C P T PP C P G P A P G P G P C P T P U P C P G P A P G P G P C P T P G P A P G P G P C P T P C P C P G Uracil DNA glycosylase AP endonuclease DNA polymerase I DNA ligase

14. 5 Methyl Cytosine Deamination Easily recognized and corrected Easily recognized and corrected What about 5-methyl cytosine? What about 5-methyl cytosine? Is there a problem? Is there a problem? Always remove T from a GT pair Always remove T from a GT pair ?

15. Deamination of Cytosine and 5- methylcytosine -------------------------------------------------------------------------------

16. Induced Mutations Caused by exposure to a mutagen Caused by exposure to a mutagen Causes Causes Exposure to base analogs Exposure to base analogs Chemical mutagens Chemical mutagens Intercalating agents Intercalating agents Uv- radiation Uv- radiation Transposable elements Transposable elements Mutator genes Mutator genes

17. Exposure to Bases Analogs Base analogs— substances that are similar to and can substitute for standard bases Base analogs— substances that are similar to and can substitute for standard bases Examples—AZT, 5- bromouracil (5-BU) and 2-aminopurine (2-AP) Examples—AZT, 5- bromouracil (5-BU) and 2-aminopurine (2-AP)

18. 5 Bromouracil The Problem: 5 bromouracil assumes the enol form at a much higher frequency than T The Problem: 5 bromouracil assumes the enol form at a much higher frequency than T  if it replaces T, will probably get a mutation due to tautomerization during replication  if it replaces T, will probably get a mutation due to tautomerization during replication Result: A·T  G·C Result: A·T  G·C A·T Replication in presence of BrU A·BrU Tautomeric shift A·BrU * Replication A·T + G·BrU * Replication A·T + A·T + G·BrU * + GC THE PROCESS