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ENVR 740
CHEMICAL CARCINOGENESIS
Instructor: Avram Gold
Office: Rosenau 157
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/2010spring/envr/740/001/
TEXTS
MOLECULAR BIOLOGY
J. E. Krebs, et al. Lewin’s Genes X, Jones and Bartlett, 2011. Not yet in HSL
B. Lewin, Genes IX, Jones and Bartlett, 2008. CALL NUMBER: QH 430 L672g
B. Lewin, Genes VIII, Pearson Prentice Hall 2004. CALL NUMBER: QH430 .L4 2004
E.C. Friedberg, G.C. Walker, W. Siede, R.D. Wood, R.A. Schultz, T. Ellenberger, DNA Repair and Mutagenesis, 2nd Ed. ASM Press
CALL NUMBER: QH467 F753 2005 (?) (Zoology Library)
J.L. Van Lancker, Apoptosis, Genomic Integrity and Cancer, Jones and Bartlett, 2006 CALL NUMBER: QU 375 V217a 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, Nature Reviews. Cancer, Cancer Research, Carcinogenesis, Chemical Research in Toxicology, Mutation Research, Cell
Syllabus, ENVR 740, Spring 2010
Introduction, chemistry overview, DNA structure.
Jan. 12, 14, 19
Class lectures and Genes IX, Ch. 1, Sec. 1.5 – 1.17
Thermodynamics
Jan. 21, 26
Class notes or Biochem text
DNA replication
Jan. 28, Feb. 2
Genes IX Ch. 15, Ch. 18
Transcriptional process
Feb. 4, 9
Genes IX, Ch. 11 (prokaryotic), Ch. 24 (eukaryotic),
Transcription/translation
Feb. 11
Genes IX, Ch. 2 Sec. 2.8 – 2.13, Ch. 3 (mRNA + processing, rRNA, tRNA), Ch. 8
Transcriptional control
Feb. 16, 18
Genes IX, Ch. 12, 13, Ch. 25 (eukaryotic promoters and enhancers)
Repair (non-enzymatic)
Feb. 23,
Genes IX, Ch. 9, Sec. 9.12 – 9.14 (suppressors)
Repair (enzymatic)
Feb. 25, Mar. 2, 4
Genes IX, Ch. 20
Signal transduction; Ras oncoproteins
[Spring break, Mar. 6-16], Mar. 16, 18
Genes VIII, Ch. 28, sec.28.1; sec. 28.5- 28.13 general; sec. 28.14-28.17 Ras
pathway; also DNA Repair & Mutagenesis, part IV
Cell cycle regulation
Mar. 23, 25
Apoptosis
Mar. 30
Genes VIII, Ch. 29, sec. 29.1-29.18
Oncogenes/tumor suppressors
Apr. 1, 6
Activation of chemical carcinogens
Apr. 8
Genes VIII, Ch. 29, sec. 29.25-29.30
P450 polymorphisms
April 13, 15
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)
DNA adducts, structure and activity
April 20, 22
Readings in current literature
Oxidative stress
April 27
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.
CANCER: in vivo process related to cell transformation in vitro
CHARACTERISTICS COMMON TO CANCER CELLS AND TRANSFORMED CELLS
loss of density-dependent growth regulation
tumor
loss of anchorage dependence
metastasis
CHARACTERISTICS UNIQUE TO CANCER CELLS
penetration of blood vessel walls by matrix metalloproteinases
development of vascular blood supply (angiogenesis)
→
metastasis
109
p-bond
o
s-bond
120o
CHIRALITY
A
B
B
A
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. Functions as base by
accepting a 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,
hydrophobic
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 monomeric 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 (x is very small)
[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
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
pol α, 5'3' synthesis but no 3'5' exonuclease capability
pol β, 5'3' synthesis with no 3'5' exonuclease capability
pol δ, 5'3' synthesis + 3'5' exonuclease capability
pol ε, 5'3' synthesis + 3'5' exonuclease capability
pol γ, 5'3' synthesis + 3'5' exonuclease capability
pol 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
3'-OH
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 and Dna2 (5 3 exonuclease)
DNA ligase I
RPA (replication protein A)
MCM (minichromosome maintenance)
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
Model illustrating how Cdt1 and geminin limit DNA replication to exactly one
round per cell cycle
The origin-recognition complex (ORC) remains bound throughout the cell cycle. During mitosis Cdt1 is
sequestered by geminin; upon exit from metaphase, geminin is degraded, releasing Cdt1. Cdt1 and
Cdc6 bind to DNA, allowing the mini-chromosome maintenance (MCM) complex to bind to DNA during
G1 phase, thereby 'licensing' DNA for a single round of replication. The MCM complex, Cdt1 and
possibly Cdc6 are displaced from DNA during S phase. Newly synthesized geminin binds to displaced
Cdt1 during S, G2 and M phases, preventing re-licensing of DNA within the same cell cycle.
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
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
stick
ribbon
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
protein synthesis
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
s (32-90 kD)
promoter specificity
ω(10 kD)
enzyme assembly
2a′ωs= 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
2-D REPRESENTATION OF E. coli RNA polymerase
–10 consensus sequence
downstream – direction of transcription
NTP access channel
“Hand” convention for representation of catalytic unit of polymerases
active site
-1
coding
anticoding
-17
thumb
N-terminal
fingers
palm
intrinsic prokaryotic terminator sequences
RNA Pol II
terminator
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.
Structure and binding of tetrameric lac repressor protein
a
b
NH2
N
N
N
N
O
O
O
P
O
O
OH
cyclic AMP
(cAMP)