2. Ensuring DNA Integrity Redundancy inherent in structureRedundancy inherent in structure DNA repair enzymologyDNA repair enzymology High precision in ReplicationHigh precision in Replication

3. DNA Polymerase III 10 protein subunits Restrictions on Nucleotide Addition –Copies only single stranded DNA –Can add only to pre-existing chains –Adds only in the 5’ to 3’ direction Proofreading –3’ to 5’ Exonuclease

4. Proofreading: 1. Mismatch Detected 2. 3’-5’ Exonuclease 3. 5’-3’ Polymerase

5. Spontaneous Mutations Forward vs. Reverse Mutations Range: 10 -4 to 10 -8 /Gene/Generation General Trends Mutations Affecting Phenotype Rare Genes Mutate at Different Rates Forward Rate > Reverse Rate

6. Bacterial Resistance to Bacteriophage Observations: 1. Most Bacteria are sensitive to Bacteriophage 2. If a culture of sensitive bacteria are spread on plate containing bacteriophage, no colonies grow. 3. Exception: a few colonies do grow, therefore they are resistant to bacteriophage. Assumption: A mutation occurred that makes those bacteria resistant. Two Hypotheses: 1. The mutation arises in response to the bacteriophage. or 2. A few bacteria already have the mutation prior to being subjected to the bacteriophage.

7. Expectation: Similar numbers of resistant colonies Expectation: Fluctuation in numbers of resistant colonies Add Selective Agent Fig. 6.4

8. Results: Culture Number# resistant colonies1 203 40 50 65 70 8107 90 1064

9. THE CAT SAW THE DOG Base Substitution THE BAT SAW THE DOG THE CAT SAW THE HOG THE CAT SAT THE DOG Insertion THE CMA TSA WTH EDO G Deletion THE ATS AWT HED OG

10. Fig. 6.6

11. Depurination Deamination Fig. 6.6

12. Excision Repair Fig. 6.7

13. Base Analogs Alkylating Agents Key Point: Chemical mutagens change the nature of the complementary base pairing Fig. 6.11

14. Perform a Complementation Test! ab “Fail to Complement”

15. Complementation Table Fig. 6.13

16. Benzer’s Fine Structure Mapping Why T4 Bacteriophage? Produce millions of progeny in a dayProduce millions of progeny in a day rII - mutationrII - mutation –1000s of mutant alleles available –Unique phenotype rII - plaquesrII - plaques rII - cannot lyse a specific bacterial strainrII - cannot lyse a specific bacterial strain –Can detect 1 recombinant/10 9 progeny

17. a1a1 + +a2a2 X + a2a2 a1a1 +

18. Gene Structure Conclusions Mutations can be order linearly Genes can be divided internally Fig. 6.16

19. Fig. 6.17

20. X-Ray Fig. 6.18

22. abcd Enz.1Enz.2Enz.3 Mutant Cannot GrowCan Grow Enzyme 1a b, c, d Enzyme 2 a, b c, d Enzyme 3 a, b, cd

23. Arginine Arg-H enzyme Argino- succinate Arg-G enzyme Citrulline Arg-F enzyme Ornithine Arg-E enzyme Fig. 6.18

24. NH 2 ---CHR---COOH H NH R C H COH O Amino Group Carboxylic Acid Side Chain

25. Fig. 6.19

26. Primary Structure Secondary Structure Tertiary Structure Fig. 6.21

27. Fig. 6.22