ENVR 740CHEMICAL CARCINOGENESIS

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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 golda_at_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/2007spring/envr/230/001/
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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)
gene mutation
integration into host DNA
v
-oncogene activation
c
-oncogene activation
mutant protein
gain/loss of protein function
altered cell biochemistry
cell transformation
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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.
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FUNCTIONAL GROUPS      
 
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WATER LATTICE
??
?-
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Polar covalent bonds
zwitterion
Ionic molecule in water lattice
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Amino Acid Residues and Codes
general amino acid
a-carbon
Optical configuration of natural amino acids l
(? S)
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HORSERADISH PEROXIDASE C chain
a
ß-sheet
a-helix
Cys 11-Cys91
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Hoogsteen pairing
The orthogonal x,y,z reference frame of the
pyrimidinepurinepyrimidine base triplet. The
y-axis is roughly parallel to the vector
connecting pyrimidine C6 and purine C8 of the TA
Watson-Crick base pair.
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minor groove
major groove
B-DNA
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Z-DNA
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H-bonding edge
anti
syn
Orientation of base around glycosydic linkage
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Hoogsteen-like pairing with modified dGuo in syn
orientation
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Common conventional representations of DNA
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EQUATIONS FOR THERMODYNAMICS   H enthalpy E
internal energy P pressure V volume Change
in enthalpy ?H ?E P ?V   S
entropy   Change in free energy ?G ?H
T?S   For the reaction as written     ?G lt 0,
spontaneous ?G gt 0, not spontaneous- work must be
put into the system to drive it in the forward
direction ?G 0, the system is in
equilibrium   K equilibrium constant, ratio of
concentrations of products to reactants     ?G
?Go RTln K R gas constant ( 1.98
cal/mole-oK 0.00198 kcal/mole-oK) T in oK ?Go
SGoproducts - SGoreactants at Pstd 1
atm, Tstd 25o C (biochem.) or 0o C (physical
chem.)   At equilibrium, ?G 0, the expression
becomes 0 ?Go RTln K or ?Go -RT ln
K Superscript o is dropped, the relationship
written as ?G -RT ln K

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Dinucleotide from 5?-deoxynucleotide phosphates
Q What is the equilibrium constant for the
formation of a dinucleotide from 5?-phosphates?
  ?G -RT ln K ?G 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-dNp-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   x55.6/1 x 10-31 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  
Q What is the equilibrium concentration of
dinucleotide from a 1 x 10-3 M initial
concentration?
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ATP H2O ADP Pi
?G -7 kcal/mole
  ADP adenosine diphosphate Pi inorganic
phosphate group ATP is sometimes written as ADPP
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 ?G lt 0   p-dN' p3-dN
p-dN'-p-dN p-p ?G 0.5 kcal/mole   p-p
H2O 2Pi ?G -7 kcal/mole p-dN'
p3-dN H2O   p-dN'-p-dN 2Pi
?G (0.5 - 7.0)kcal/mole
-6.5 kcal/mole  
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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
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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.
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Effect of enzyme on ?G
?G
?G
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5 3 addition



P-P
H2O
2Pi


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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
a, 5'3' synthesis but no 3'5' exonuclease ß,
5'3' synthesis with no 3'5' exonuclease d,
5'3' synthesis 3'5' exonuclease e, 5'3'
synthesis 3'5' exonuclease ?, 5'3' synthesis
3'5' exonuclease a -e are located in the
nucleus, and ? in mitochondria. a initiates
strand synthesis, d is responsible for strand
extension, e and ß are involved in repair while
? is responsible for replication of mitochondrial
DNA
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SSBs
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1
4
ß-clamp
t
2
3
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Some Eukaryotic Replication Proteins
DNA pol a RNA priming short 3 4 base DNA
extension (iDNA i initiation) DNA pol
d Strand extension PCNA (proliferating cell
nuclear antigen) Processivity
(equivalent function to ß-clamp) RFC (replication
factor C) Loads pol d and PCNA at end of
iDNA FEN1, Dna2 (5 3 exonuclease)
Removal of RNA primer DNA ligase I Seal
nicks RPA Single strand binding
proteins MCM Helicase function
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MODEL OF EUKARYOTIC REPLICATION FORK
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prokaryotic origin of replication
                                               
                                                  
                                                  

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fully methylated
hemi-methylated
A
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Autonomously replicating sequence ARS
of origin function
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geminin
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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
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DNA-RNA distinctions
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5'NNN3'
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acceptor arm
T?C
D arm
extra arm
anticodon
Amino acid
T?C arm
D arm
anticodon arm
anticodon
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O
H
N
N
H
C
O
(
y
)
p
s
e
u
d
o
u
r
i
d
i
n
e


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3?-terminus
Yeast phe tRNA (not charged with aa)
5?-terminus
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Wobble hypothesis rules for codon/anticodon
pairing
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Genes VIII, Fig. 6.2
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Genes VIII, Fig. 6.7
Genes VIII, Fig. 6.3
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EUKARYOTIC mRNA PROCESSING
PROKARYOTIC mRNA/PROTEIN SYNTHESIS
Genes VIII, Fig. 5.17
Genes VIII, Fig. 5.13
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5?-CAPPING OF EUKARYOTIC mRNA
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introns
exon
exon
exon
splice
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STEM LOOP
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Subunits of prokaryotic RNA polymerase
Catalytic core
2abb's holoenzyme
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coding strand
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intrinsic prokaryotic terminator sequences
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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-a
cting control elements acting only on coding
sequences directly down-stream. structural genes
code for proteins. regulator genes code for
products that are involved in regulating the
expression of other genes.
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hinge helix-turn-helix
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IPTG (isopropylthioglucose)
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truncation at hinge
truncation at hinge
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Tetramer, with two of the tetrameric units
selected
truncation at point of hinge attachment
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Lac repressor dimer bound to operator
Headpiece (hinge HTH motif)
hinge
B. Rotated 90o around core axis
A. Looking down DNA helix
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Contrast inducer-bound and active lac repressor
Lac repressor ONPF truncated at oligomerization
domain
Lac repressor IPTG truncated at hinge.
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