ENVR 230 CHEMICAL CARCINOGENESIS Instructor: Avram Gold Office: McGavran-Greenberg 4114C Office phone: 6 7304 Lab: McGa

1. ENVR 230 CHEMICAL CARCINOGENESIS Instructor: Avram Gold Office: McGavran-Greenberg 4114C Office phone: 6 7304 Lab: McGavran-Greenberg 3221E Lab phone: 6 7325 e-mail: golda@email.unc.edu Grading 2 exams: final, 60%; midterm, 30%; homework + class participation 10%. Four problem sets during semester- more if current literature section is larger. Course web site To be established at: http//www.unc.edu/courses/2004spring/envr/230/001/

2. TEXTS MOLECULAR BIOLOGY B. Lewin, Genes VIII, Pearson Prentice Hall 2004. CALL NUMBER: Availability estimated mid-January  BASIC BIOCHEMISTYRY 1. J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (2nd ed.) Freeman and Co. 1995. CALL NUMBER QH581.2 .D223m 1995 2. B. Alberts, D. Bray. J. Lewis, M. Raff, K. Roberts, J.D. Watson Molecular Biology of the Cell (2nd ed.) Garland Publishing 1994. CALL NUMBER QH581.2 .M718 1994 3. Christopher K. Mathews, K.E. van Holde, Kevin G. Ahern, Biochemistry San Francisco, CA : Benjamin Cummings, 2000. CALL NUMBER: QU 4 M4294b 2000 4. Richard I. Gumport ... [et al.], Student's Companion to Stryer's Biochemistry New York : W.H. Freeman, 1990. CALL NUMBER: QP514.2 .S662 1990 JOURNALS Cancer Research Carcinogenesis Chemical Research in Toxicology Mutation Research Nature Science

3. Introduction, chemistry overview, DNA structure. Jan. 11, 13, 18 Genes VIII, Ch. 1-2 through sec. 2.8 Ch. 30, sec. 30.1-30.2 Thermodynamics Jan. 20, 25 Class notes or Biochem text DNA replication Jan. 27, Feb. 1 Ch. 13, sec. 13.1-13.6, 13.8; Ch. 14 Transcriptional process Feb. 3, 8 Ch. 9, sec. 9.1-9.17, 9.20; Ch. 21, sec. 21.1-21.20 (promoters and enhancers) Transcription/translation Feb. 10 Ch. 5 (mRNA + processing, rRNA, tRNA); Ch. 6, sec. 6.1, 6.2-6.8, 6.14, 6.15 other sec. optional); Ch. 7, sec. 7.1, 7.2, 7.4, 7.5, other optional) Transcriptional control Feb. 15, 17 Ch. 11, 12 entirety Repair (non-enzymatic) Feb. 22, Ch. 7, sec. 7.11-7.18 (suppressors) Repair (enzymatic) Feb. 24, Mar. 1 Ch. 15, sec. 15.1-15.19 optional, details of recombination; sec. 15.20-15.30 Signal transduction; Ras oncoproteins March 3, 8 Ch. 28, sec.28.1; sec. 28.5- 28.13 general; sec. 28.14-28.17 Ras pathway Cell cycle regulation Mar. 10 Ch. 29, sec. 29.1-29.18 Spring break, Mar. 11-21 Cell cycle regulation Mar. 22 Apoptosis Mar. 24 Ch. 29, sec. 29.25-29.30 Oncogenes/tumor suppressors Mar. 29, 31 Ch. 30, sec. 30.3, sec. 30.6-30.11, (sec. 30.14-30.18 optional), 30.19-30.23, (sec. 30.25 and 30.26 optional) Activation of chemical carcinogens Apr. 5, Readings in current literature P450 polymorphisms April 7, 12 DNA adducts, structure and activity April 14, 19 Oxidative stress April 21, 26

4. processing of lesions by repair or by replication apparatus PATHWAYS TO CELL TRANSFORMATION CHEMICAL metabolic activation of exogenous chemicals endogenous generation of reactive species interaction with DNA and generation VIRAL of DNA lesions infection with transforming virus: DNA or RNA (retrovirus) integration into host DNA gene mutation v -oncogene activation c -oncogene activation mutant protein gain/loss of protein function altered cell biochemistry cell transformation

5. CHARACTERISTICS OF TRANSFORMED CELLS (1) Immortalization and aneuploidy. (2) Unrestricted growth; loss of density-dependent regulation (or contact inhibition), formation of foci. (3) Loss of anchorage dependence for growth. (4) Requirement for growth factor containing serum to sustain growth is absent or reduced. (5) Cytoskeletal changes. (6) Dedifferentiation - loss of cell function. (7) Tumorigenic when injected into syngenetic host.

7. -OH hydroxy Alcohol, e.g., ethanol, methanol. Hydroxy groups impart solubility in water. -C(=O)OH carboxyl Organic (carboxylic) acid, e.g., acetic acid. Carboxyl group is acidic by ionization releasing a proton. Presence also enhances water solubility. -NH2 amino Base, by virtue of donation of unshared electrons of trivalent nitrogen. Acceptor of proton from ionized organic or mineral acids. FUNCTIONAL GROUPS

8. WATER LATTICE  -

9. R-CH-CO2- NH3+ Cδ+-Oδ- Oδ--Hδ+ Nδ--Hδ+ Polar covalent bonds zwitterion Ionic molecule in water lattice

10. CARBON TETRACHLORIDE IS NON-POLAR C l C l d - d + d - d + C d + d - d + C l d C l -

12. neutral, hydrophobic neutral, polar bases and acids Amino Acid Residues and Codes general amino acid a-carbon Optical configuration of natural amino acids: l ( S)

14. HORSERADISH PEROXIDASE C chain a β-sheet α-helix Cys 11-Cys91

17. Hoogsteen pairing The orthogonal x,y,z reference frame of the pyrimidine·purine+pyrimidine base triplet. The y-axis is roughly parallel to the vector connecting pyrimidine C6 and purine C8 of the T·A Watson-Crick base pair.

18. minor groove major groove B-DNA

19. Z-DNA

20. H-bonding edge syn anti Orientation of base around glycosydic linkage

21. Hoogsteen-like pairing with modified dGuo in syn orientation

23. Common conventional representations of DNA

24. A + B A-B + H2O

25. EQUATIONS FOR THERMODYNAMICS H ≡ enthalpy E ≡ internal energy P ≡ pressure V ≡ volume Change in enthalpy: ΔH = ΔE + P ΔV S ≡ entropy Change in free energy: ΔG = ΔH – TΔS For the reaction as written: ΔG < 0, spontaneous ΔG > 0, not spontaneous- work must be put into the system to drive it in the forward direction ΔG = 0, the system is in equilibrium K ≡ equilibrium constant, ratio of concentrations of products to reactants: ΔG = ΔGo + RTln K R ≡ gas constant (= 1.98 cal/mole-oK = 0.00198 kcal/mole-oK) T in oK ΔGo = ΣGoproducts - ΣGoreactants at Pstd = 1 atm, Tstd = 25o C (biochem.) or 0o C (physical chem.) At equilibrium, ΔG = 0, the expression becomes: 0 = ΔGo + RTln K or ΔGo = -RT ln K Superscript “o” is dropped, the relationship written as: ΔG = -RT ln K A-B + H2O A + B

26. p-dN + p-dN¢ p-dN-p-dN¢ + H2O ΔG = +6 kcal/mole Dinucleotide from 5-deoxynucleotide phosphates Q: What is the equilibrium constant for the formation of a dinucleotide from 5-phosphates? ΔG = -RT ln K ΔG = +6 kcal/mole R = 0.00198 kcal/mole-oK T = (25 + 273) o K = 298 oK 6 kcal/mole = -(0.00198kcal/mole-oK)(298 oK)ln K ln K = -6/(1.98 x 10-3)(298) = -10.2 K = e-10.2 = 3.83 x 10-5 K= 3.83 x 10-5 = [p-dN-p-dN][H2O]/[p-dN][p-dN] Initial dinucleotide concentration [p-dN-p-dN¢] = 1 x 10-3 M Virtually all the dimer will disappear; therefore, approximate the product nucleotides as [p-dN] = [p-dN¢] » 1 x 10-3 M Exact expression is [p-dN] = [p-dN¢] = (1 x 10-3 –x) [dimer] = x [H20] ≈ constant = 55.6 M [x][55.6]/[1 x 10-3][1 x 10-3] = 3.8 x 10-5 [x] = (3.8 x 10-5)(1 x 10-3)2/55.6 = 6.8 x 10-13 M Ratio of dinucleotide to mononucleotides: [p-dN-p-dN¢]/[p-dN][p-dN¢] = 6.8 x 10-7 Q: What is the equilibrium concentration of dinucleotide from a 1 x 10-3 M initial concentration?

27. ATP + H2O ADP + Pi ΔG = -7 kcal/mole ADP = adenosine diphosphate Pi = inorganic phosphate group ATP is sometimes written as ADP~P to emphasize high energy of the phosphate bond The first stage in polynucleotide synthesis is the transfer of a high-energy bond to p-dN in two steps: ATP + p-dN ADP + dNDP ATP + dNDP ADP + dNTP ΔG ~< 0 p-dN′ + p3-dN p-dN′-p-dN + p-p ΔG = +0.5 kcal/mole p-p + H2O 2Pi ΔG = -7 kcal/mole p-dN′ + p3-dN + H2O   p-dN′-p-dN + 2Pi ΔG = (+0.5 - 7.0)kcal/mole = -6.5 kcal/mole

28. Hydrolysis of phosphodiester linkage - O O O 5 ' - d N O 5 ' - d N ' P P 5’-d NMP - 3 ' - O 5'-dNMP + 5'-dN'MP 5’-d NMP - 3 ' - O OH - O - OH - O transition state transition state DG‡ DG

29. In the Kf exonuclease reaction, the 3' terminal phosphodiester linkage of a DNA oligonucleotide is cleaved by attack of water or hydroxide ion, yielding dNMP and a shortened oligonucleotide ending with a 3' hydroxyl. The most prominent structural feature of the exonuclease site is a binuclear metal center that is proposed to mediate phosphoryl transfer (Figure 1a). In enzyme-product (dNMP) complexes, a pentacoordinate metal (A) shares a ligand, Asp-355, with an octahedral metal (B).8b,c Superposition of wild-type structures bound with product onto mutant enzyme structures (lacking metal ion B) bound with oligonucleotide substrate8b,c,9 places the 3' oxygen atom (the leaving group) of the substrate within the inner coordination sphere of metal ion B (2.4 Å).8b Therefore, metal ion B is proposed to interact directly with the 3' oxygen atom in the transition state, presumably stabilizing the developing negative charge on the oxyanion leaving group. Although the two-metal-ion mechanism of Kf is thought to be a general strategy by which many protein enzymes and ribozymes catalyze phosphoryl transfer,8a,10 there is no direct biochemical evidence that the 3'-5' exonuclease employs a metal ion in this role.

30. Effect of enzyme on ΔG‡ ΔG‡ ΔG

31. THREE STAGES OF REPLICATION initiation – recognition of origin elongation – extension by replisome termination  2 pi

32. B B B B B B B B B proofreading B B B B' B' B' P P P P P P P P P P P P P P P OH OH P P P P P3 OH OH OH B' B' P3 P3 OH OH B’' P3 OH P 3’ 5’ addition proofreading ? B' B B B B B B B B B B B P P P P3 P3 P B‘’ P3 OH B P 5’ 3’ addition + + + P-P H2O 2Pi + +

33. PROKARYOTIC POLYMERASES pol I, 5'®3' synthesis + 3'®5' exonuclease, unique 5'®3' exonuclease capability. Pol I responsible for repair, since 5'®3' exonuclease activity allows pol I to extend a strand from a nick in DNA. (Nick: strand break caused by hydrolysis of phosphodiester bond.) pol II, 5'®3' synthesis + 3'®5' exonuclease, also is involved in repair. pol III, large multi-unit enzyme 5'®3' synthesis + 3'®5' exonuclease, primarily involved in strand extension during replication. EUKARYOTIC POLYMERASES α, 5'®3' synthesis but no 3'®5' exonuclease β, 5'®3' synthesis with no 3'®5' exonuclease δ, 5'®3' synthesis + 3'®5' exonuclease ε, 5'®3' synthesis + 3'®5' exonuclease γ, 5'®3' synthesis + 3'®5' exonuclease α -ε are located in the nucleus, and γ in mitochondria. α initiates strand synthesis, δ is responsible for strand extension, ε and β are involved in repair while γ is responsible for replication of mitochondrial DNA

37. 3' 5' Direction of replication fork progression

38. SSBs

39. 1 4 β-clamp τ 2 3

40. Some Eukaryotic Replication Proteins DNA pol α RNA priming + short 3 – 4 base DNA extension (iDNA; i = initiation) DNA pol δ Strand extension PCNA (proliferating cell nuclear antigen) Processivity (equivalent function to β-clamp) RFC (replication factor C) Loads pol δ and PCNA at end of iDNA MF1 (5¢® 3¢ exonuclease) Removal of RNA primer DNA ligase I Seal nicks

41. MODEL OF EUKARYOTIC REPLICATION FORK

42. 9-mers 13-mers

43. Autonomously replicating sequence: ARS % of origin function

45. Codons are represented as the mRNA coding strand. DNA not copied: sense/coding strand Double stranded DNA template DNA: antisense/anticoding strand mRNA coding strand DNA-RNA hybrid template DNA: antisense/anticoding strand

46. DNA-RNA distinctions

47. 5'NNN3'

48. Amino acid TC arm D arm anticodon arm anticodon

49. O H N N H O C ( y ) p s e u d o u r i d i n e

50. 3-terminus Yeast phe tRNA (not charged with aa) 5-terminus