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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
acarbon
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-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
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
TC
D arm
extra arm
anticodon
Amino acid
TC 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
5AGC3
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)