1. Mutation, DNA Repair and Recombination BIT 220 Chapter 14

2. Mutation Heritable change in genetic material –change in gene –change in chromo # –change in chromo structure Germinal Mutation –may be passed to progeny Somatic Mutation –NOT passed to progeny offspring

3. What causes mutation? SPONTANEOUS –No known cause –maybe be indeed a causative agent, but has not been identified INDUCED –Caused by MUTAGEN (physical or chemical agents which alter DNA) nicotine UV light drugs ionizing radiation

4. Phenotypic Effects Neutral Mutation- NO Effect on phenotype »ISOALLELES Null Allele - No functional protein Recessive Lethal - deadly null mutation in both alleles

5. Conditional Lethal Mutations Define: Lethal in one environment (restrictive condition) Viable in second environment (permissive condition) 1. Auxotrophs Mutants - can’t synthesize metabolite 2. Temperature-Sensitive mutants - grow in one T, not another T 3. Suppressor-sensitive mutants - will grow in presence of suppressor; suppressor protein compensates for mutation

6. Replica Plating Figure 14.3 Non-Selective Plates -no antibiotic Selective plates- antibiotic in medium Mutation does NOT occur in response to selective agent

7. Frameshift Mutations Base pair addition or deletion alter reading frame always result in non-functional protein

8. Forward and Reverse Mutations Figure 14.4 Forward: wild-type to mutant phenotype Reverse: 2nd mutation restores earlier phenotype; reverse occurs by back mutation or suppressor mutation Summary of mutation process: Figure 14.7 Example (sickle-cell) Figure 14.8

9. Transposons Figure 14.24 Pieces of DNA that can move from one location of genome to another No specific target site Effects: help bacteria to adapt to new environments - develop antibiotic resistance can interrupt functional genes can carry another gene sit behind promoter and upregulate expression of gene it carries variegation of color in corn

10. DNA Repair Mechanisms Enzymes constantly scan DNA looking for errors. Repair can occur during or after replication Mutations which cripple the repair system Xeroderma pimentosum can not correct UV-induced damage to DNA

11. Mechanisms of Repair A. DNA Polymerase corrects mistakes during replication 3’-5’ exonuclease activity B. Mismatch Repair wrong NT (non complementary base) added during synthesis corrected after replication differentiates between methylated strand and unmethylated C. Excision Repair Figure 14.27 and Figure 14.28 errors made from mutagens (UV rays, X rays) NUCLEASE cuts out error POLYMERASE replaces NT LIGASE seals up correction made post replication

12. Mutagenesis:Why Mutate? Native proteins are not well suited for industrial application Native proteins are not optimized for medicinal purposes Increase the efficiency of enzyme-catalyzed reactions Eliminate the need for cofactor in enzymatic reaction Change substrate binding site to increase specificity Change the thermal tolerance Change the pH stability Increase proteins resistance to proteases (purification)

13. Protein Engineering Enzymes Used in Industrial Applications amylase beer production lipase cheese production papainmeat tenderizer protease detergents Problems Conditions of process inactivate the enzyme high temperature pH ranges solvents Specific Changes at the Protein Level Add Disulfide Bonds Changing Asparagine to Other amino Acids

14. Adding Disulfide Bonds Usually found in extracellular proteins, not intracellular Cross link between chains or in chains formed by oxidation of cysteine residues connective tissue fibrin blood clots Why would we add disulfide bonds? -Artificial addition may increase stability of protein As with all engineering AVIOD active site (enzyme) XYLANASE used to treat wood pulp in paper production needs to function at high temp

15. Aspargine Changes At high T, asparagine and glutamine deaminate ammonia is released amino acids convert to aspartic acid and glutamic acid Protein may refold LOSE ACTIVITY Use Computer modeling to predict where mutation is preferred Test stability/activity/stability of enzyme in vivo/in vitro

16. Reducing Free Sulfhydryl Residues Cysteine residues may cause dimerization through intermolecular disulfide bonding Convert Cys to another amino acid reduce dimerization maintain activity of enzyme

17. Enzyme Activity and Specificity Increase enzymatic activity by increasing affinity for enzyme change sequences in substrate binding site Change substrate of enzyme

18. Other Examples tPA tissue plasminogen activator dissolves blood clots would like to decrease clearance decrease non-specific bleeding increase specificity for fibrin in blood clot Eliminate need for cofactors Serine protease are used in laundry detergents require calcium modify enzymes ability to bind calcium