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ENVR 740 CHEMICAL CARCINOGENESIS Instructor: Avram Gold Office: McGavran-Greenberg 4114C Office phone: 6 7304 Lab: McGavran-Greenberg 3221E Lab phone: 6 7325 e-mail: [email protected] 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/2007spring/envr/230/001/ TEXTS MOLECULAR BIOLOGY B. Lewin, Genes VIII, Pearson Prentice Hall 2004. (Genes IX, Jones and Bartlett due out 03/07) CALL NUMBER: QH430 .L4 2004 D. Warshawsky, J.R. Landolph, Molecular Carcinogenesis and the Molecular Biology of Human Cancer, Taylor and Francis CALL NUMBER: QZ200 M71833 2006 BASIC BIOCHEMISTYRY 1. J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (5th ed.) Freeman and Co. 2004. CALL NUMBER: QH 581.2 D223m 2004 2. B. Alberts, D. Bray. J. Lewis, M. Raff, K. Roberts, J.D. Watson Molecular Biology of the Cell (4th ed.) Garland Publishing 2002. CALL NUMBER: QH581.2 .M64 2002, reserve 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. J.M. Berg, J.L. Tymoczko, L. Stryer, Biochemistry New York : W.H. Freeman, 2006. Available from HSL: CALL NUMBER: QU 4 S928b 2002 JOURNALS Science, Nature, Cancer Research, Carcinogenesis, Chemical Research in Toxicology, Mutation Research Introduction, chemistry overview, DNA structure. Jan. 11, 16, 18 Genes VIII, Ch. 1-2 through sec. 2.8 Ch. 30, sec. 30.1-30.2 Thermodynamics Jan. 23, 25 Class notes or Biochem text DNA replication Jan. 30, Feb. 1 Ch. 13, sec. 13.1-13.6, 13.8; Ch. 14 Transcriptional process Feb. 6, 8 Ch. 9, sec. 9.1-9.17, 9.20; Ch. 21, sec. 21.1-21.20 (promoters and enhancers) Transcription/translation Feb. 13 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, 20 Ch. 11, 12 entirety Repair (non-enzymatic) Feb. 22, Ch. 7, sec. 7.11-7.18 (suppressors) Repair (enzymatic) Feb. 27, Mar. 1 Ch. 15, sec. 15.1-15.19 optional, details of recombination; sec. 15.20-15.30 Signal transduction; Ras oncoproteins Mar. 6, 8 Ch. 28, sec.28.1; sec. 28.5- 28.13 general; sec. 28.1428.17 Ras pathway Spring break, Mar. 9-19 Cell cycle regulation Mar. 20 Ch. 29, sec. 29.1-29.18 Cell cycle regulation Mar. 22 Apoptosis Mar. 27 Ch. 29, sec. 29.25-29.30 Oncogenes/tumor suppressors Mar. 29, Apr. 3 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 10, 12 DNA adducts, structure and activity April 17, 19 Oxidative stress April 24, 26 PATHWAYS TO CELL TRANSFORMATION CHEMICAL metabolic activation of exogenous chemicals endogenous generation of reactive species interaction with DNA and generation of DNA lesions VIRAL infection with transforming virus: DNA or RNA (retrovirus) processing of lesions by repair or by replication apparatus integration into host DNA v-oncogene activation gene mutation c-oncogene activation mutant protein gain/loss of protein function altered cell biochemistry cell transformation 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. bond 109o bond 120o CHIRALITY A A B D A B B C B A C D cis enantiomers BOND ENERGIES 83 Kcal/mole, C-C (single) bond 150 Kcal/mole, C=C (double) bond trans FUNCTIONAL GROUPS -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. WATER LATTICE H O H O H H O H O H O H- H O H H O H O O H O H O O H O O O H H H H H H O H H H H H O O H H H H H H O H H H H H H H H O H O H H Polar covalent bonds R-CH-CO2 NH3+ - Cδ+-Oδ- Oδ--Hδ+ Nδ--Hδ+ zwitterion Ionic molecule in water lattice H O H O H O O O H O H H H H H H H O H H O H H H O H H O H O H H O H O H O H H O O O H H H H H O H H H H H H H O H O H H CARBON TETRACHLORIDE IS NON-POLAR Cl- + + + - Cl Cl C + - Cl Hydrdogen bonds are directional: linear provides maximum overlap N H O H O O O R R O H R= alanine Ala, A Valine Val, V Leucine Leu, L CH3 CH3 CH3 Tryptophan Trp, W CH3 CH2 CH3 CH3 Phenylalanine Phe, F Amino Acid Residues and Codes Isoleucine Ile, I CH3 Methionine Met, M neutral, hydropho bic Proline Pro, P CO2H CH2 NH S CH2 HN CH3 general amino acid H2C CH2 O Glycine Gly, G Serine Ser, S H Threonine Thr, T CH2 OH HO Tyrosine Tyr, Y H2N * CH R CH2 CH3 acarbon neutral, polar OH Cysteine Cys, C Asparagine Asn, N H2C CH2 NH2 O H2C O Lysine Lys, K Arginine Arg, R Histidine His, H CH2 NH2 Aspartic acid Asp, D H2C O N HN H2N HN NH2 Optical configuration of natural amino acids: l ( S) Glutamine Glu, Q H2C SH NH2 OH Glutamic acid Glu, E CH2 H2C HO O HO bases and acids Bend in backbone introduced by proline O N H O Distant regions brought into juxtaposition by disulfide bond R O O R NH NH S O S NH NH R O NH R HORSERADISH PEROXIDASE C chain a β-sheet α-helix Cys 11-Cys91 purines pyrimidines NH2 O guanine, Gua or G { N HN H2N N HO O HO P O O [ O N O [guanylic acid] [deoxycytidylic acid ] NH2 O adenine, Ade or A { [ N ] O O N HO O HO P - O O OH [ ] O N deoxythymidine acid, dThyd [deoxyadenylic acid ] [deoxythymidylic acid] deoxynucleoside base base + deoxyribose 4 6 7 3 1 3 2 8 9 5 1 1' 2' 4' 2' 3' 6 1' 5' 3' } thymine, T OH deoxyadenosine, dAdo nucleobase 4' CH3 HN N N OHO HO P O O 5' ] [ deoxyguanosine, dGuo 2 O cytosine, C N HO OH O HO P O O deoxycytidine, dCyd OH ] } N numbering convention deoxynucleotide (nucleic acid) base + deoxyribose-5'-phosphate 3’ 5’ phosphodiester { 3’ bond 5 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. minor groove major groove B-DNA Z-DNA H-bonding edge syn anti Orientation of base around glycosydic linkage Hoogsteen-like pairing with modified dGuo in syn orientation N N O N H N H2N H O N N O HN N NH2 5' 3' A T C A G A T A G T C T 3' 5' B P B P B P B P OH Common conventional representations of DNA A+B forward backward A-B + H2O 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: A+B forward backward A-B + H2O 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: K [ A B][ H 2O] [ A][ B] 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 Superscript “o” is dropped, the relationship written as: ΔG = -RT ln K ΔGo = -RT ln K Dinucleotide from 5-deoxynucleotide phosphates Q: What is the equilibrium constant for the formation of a dinucleotide from 5-phosphates? p-dN + p-dN p-dN-p-dN + H2O ΔG = +6 kcal/mole ΔG = -RT ln K ΔG = +6 kcal/mole o R = 0.00198 kcal/mole- K 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 Q: What is the equilibrium concentration of dinucleotide from a 1 x 10-3 M initial concentration? K= 3.83 x 10-5 = [p-dN-p-dN][H2O]/[p-dN][p-dN] Initial dinucleotide concentration [p-dN-p-dN1 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 NH2 ATP + H2O ΔG = -7 kcal/mole ADP + Pi N N N N ADP = adenosine diphosphate O - P O Pi = inorganic phosphate group ATP is sometimes written as ADP~P to emphasize high energy of the phosphate bond O O O- 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 ΔG = -7 kcal/mole 2Pi p-dN′ + p3-dN + H2O p-dN′-p-dN + 2Pi ΔG = (+0.5 - 7.0)kcal/mole = -6.5 kcal/mole P O- O O P O- O O OH OH Hydrolysis of phosphodiester linkage - O O O 5'-dN O 5'-dN' 5’-dNMP-3'-O P P OH 5’-dNMP-3'-O - O -OH 5'-dNMP + - O transition state G transition state G‡ reactants G products reaction coordinate 5'-dN'MP 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. Effect of enzyme on ΔG‡ G ΔG‡ reactants ΔG products reaction coordinate THREE STAGES OF REPLICATION initiation – recognition of origin elongation – extension by replisome termination 2 pi B’' 5’ P3 3’ addition B P B P B P B P OH + B' P3 B P OH B P B B P P proofreading B' P B P OH B P B P B' B P OH + OH P + P-P H2O 2Pi B‘’ 3’ 5’ addition B' P3 B + OH P3 P3 B P B P B' B P P3 B P B P proofreading B P B P OH B' P3 B + OH P B P B P B P ? OH 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 5' Direction of replication fork progression 3' SSBs 1 4 β-clamp τ 2 3 Some Eukaryotic Replication Proteins DNA pol α DNA pol δ PCNA (proliferating cell nuclear antigen) RFC (replication factor C) FEN1, Dna2 (5 3 exonuclease) DNA ligase I RPA MCM RNA priming + short 3 – 4 base DNA extension (iDNA; i = initiation) Strand extension Processivity (equivalent function to β-clamp) Loads pol δ and PCNA at end of iDNA Removal of RNA primer Seal nicks Single strand binding proteins Helicase function MODEL OF EUKARYOTIC REPLICATION FORK prokaryotic origin of replication control of replication at prokaryotic origins G (*A) T C G (*A) T C C T (*A) G C T (A) G parent duplex parent + daughter duplex fully methylated hemi-methylated CH3 HN N N *A = N N H N6-MeAde Autonomously replicating sequence: ARS % of origin function Mcm Mcm geminin 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 DNA-RNA distinctions DNA O HO RNA B O HO B 2' OH OHOH deoxyribose ribose O CH3 HN O O NH thymine HN O NH uracil 5'NNN3' U C A G U UUU Phe UUC UUA Leu UUG CUU CUC Leu CUA CUG AUU AUC Ile AUA AUG Met GUU GUC Val GUA GUG C UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala A UAU Tyr UAC UAA STOP UA G CAU His CAC CAA Gln CA G AAU Asn AAC AAA Lys AAG GA U Asp GA C GAA Glu GA G G UGU Cys UGC UGA STOP UGG Trp CGU CGC Arg CGA CGG AGU Ser AGC AGA Arg AGG GGU GGC Gly GGA GGG acceptor arm TC D arm extra arm anticodon Amino acid TC arm D arm anticodon arm anticodon O HN O NH C pseudouridine O HN O CH2 N CH2 dihydrouridine D Yeast phe tRNA (not charged with aa) 3-terminus 5-terminus 1 2 3 codon 5AGC3 anticodon 3 UCG5 3 2 1 codon AGC anticodon GCU Wobble hypothesis: rules for codon/anticodon pairing U in position 1 of the anticodon pairs with A or G in position 3 of codon C G only A U only G C or U Genes VIII, Fig. 6.2 Genes VIII, Fig. 6.7 Genes VIII, Fig. 6.3 PROKARYOTIC mRNA/PROTEIN SYNTHESIS EUKARYOTIC mRNA PROCESSING Genes VIII, Fig. 5.17 Genes VIII, Fig. 5.13 5-CAPPING OF EUKARYOTIC mRNA introns exon exon splice exon STEM LOOP N N N N N N N N N N A C U C G GCUCANNNNNNNNNNUGAGC Subunits of prokaryotic RNA polymerase Subunit (molecular weight) Function 2 x a (40 kD) enzyme assembly, promoter recognition (155 kD) catalytic center (160 kD) catalytic center (32-90 kD) promoter specificity 2a′= holoenzyme upstream, -n start point downstream, +n +1 3' 5' coding strand -10 consensus sequence T80 A95 T45 A60 A50 T96 -35 consensus sequence T82 T84G78A65C54A45 intrinsic prokaryotic terminator sequences operon: Coding region of structural genes and the elements that control their expression. genes: elements of DNA that code for diffusible products. trans-acting: control elements acting at sites distant from site of transcription. cis-acting: control elements acting only on coding sequences directly downstream. structural genes: code for proteins. regulator genes: code for products that are involved in regulating the expression of other genes. hinge + helix-turn-helix IPTG (isopropylthioglucose) OH HO HO OH CH2 O S truncation at hinge truncation at hinge Tetramer, with two of the tetrameric units selected truncation at point of hinge attachment Lac repressor dimer bound to operator Headpiece (hinge + HTH motif) hinge anti-inducer A. Looking down DNA helix B. Rotated 90o around core axis o-nitrophenylfructose (ONPF) Contrast inducer-bound and active lac repressor Lac repressor + ONPF truncated at oligomerization domain Lac repressor + IPTG truncated at hinge. NH2 N N N N O O O P O O OH cyclic AMP (cAMP)