ENVR 740 CHEMICAL CARCINOGENESIS

Instructor: Avram Gold

Office: Rosenau 157

Office phone: 6 7304

Lab: McGavran-Greenberg 3221E

Lab phone: 6 7325

e-mail: golda@email.unc.eduGrading

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 PressCALL 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

Oxidative stressApril 27

Readings in current literatureDNA adducts, structure and activityApril 20, 22

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)

P450 polymorphismsApril 13, 15

Genes VIII, Ch. 29, sec. 29.25-29.30Activation of chemical carcinogensApr. 8

Oncogenes/tumor suppressorsApr. 1, 6

Genes VIII, Ch. 29, sec. 29.1-29.18ApoptosisMar. 30

Cell cycle regulationMar. 23, 25

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

Signal transduction; Ras oncoproteins[Spring break, Mar. 6-16], Mar. 16, 18

Genes IX, Ch. 20Repair (enzymatic)Feb. 25, Mar. 2, 4

Genes IX, Ch. 9, Sec. 9.12 – 9.14 (suppressors)Repair (non-enzymatic)Feb. 23,

Genes IX, Ch. 12, 13, Ch. 25 (eukaryotic promoters and enhancers)Transcriptional controlFeb. 16, 18

Genes IX, Ch. 2 Sec. 2.8 – 2.13, Ch. 3 (mRNA + processing, rRNA, tRNA), Ch. 8Transcription/translationFeb. 11

Genes IX, Ch. 11 (prokaryotic), Ch. 24 (eukaryotic), Transcriptional processFeb. 4, 9

Genes IX Ch. 15, Ch. 18DNA replicationJan. 28, Feb. 2

Class notes or Biochem textThermodynamicsJan. 21, 26

Class lectures and Genes IX, Ch. 1, Sec. 1.5 – 1.17Introduction, chemistry overview, DNA structure.Jan. 12, 14, 19

Syllabus, ENVR 740, Spring 2010

PATHWAYS TO CELL TRANSFORMATION

processing of lesions by repair orby replication apparatus

mutant proteingain/loss of protein function

altered cell biochemistry

cell transformation

infection with transformingvirus: DNA or RNA (retrovirus)

metabolic activation of exogenous chemicalsendogenous generation of reactive species

CHEMICAL

VIRALinteraction with DNA and generationof DNA lesions

gene mutationc-oncogene activation

integration into host DNAv-oncogene activation

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 → metastasis

development of vascular blood supply (angiogenesis)

109o

B

A

BA

cis trans

bond

bond

120o

CHIRALITY

enantiomers

BOND ENERGIES

83 Kcal/mole, C-C (single) bond150 Kcal/mole, C=C (double) bond

A

C B

DA

CB

D

 

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.

 

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

HH

OH

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

WATER LATTICE

-

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

HH

OH

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

Cδ+-Oδ- Oδ--Hδ+ Nδ--Hδ+

Polar covalent bonds

Ionic molecule in water lattice

R-CH-CO2-

NH3+

zwitterion

C

Cl Cl

ClCl

-+-

+

-+

-

+

CARBON TETRACHLORIDE IS NON-POLAR

N H O

O

ORH

O

O RH

Hydrdogen bonds are directional: linear provides maximum overlap

neutral, hydrophobic

neutral, polar

bases and acids

R=

GlycineGly, G

SerineSer, S

ThreonineThr, T

TyrosineTyr, Y

CH2

OHCH3HO

OH

CH2H

H2CSH

H2C

ONH2

CH2

H2C

ONH2

CysteineCys, C

AsparagineAsn, N

GlutamineGlu, Q

H2NHN

HNNH2

NNH2

CH2 H2C

HOO

CH2

H2C

HOO

LysineLys, K

ArginineArg, R

HistidineHis, H

Glutamic acidGlu, E

Aspartic acidAsp, D

CH3

CH3 CH3

CH3 CH3

CH2

CH3

CH3

alanineAla, A

ValineVal, V

LeucineLeu, L

IsoleucineIle, I

TryptophanTrp, W

PhenylalaninePhe, F

MethionineMet, M

ProlinePro, P

NH

CH2

SCH3

HN

H2C CH2

CH2

CO2H

Amino Acid Residues and Codes

OHCHH2N

O

R

*

general amino acid

carbon

Optical configuration of natural amino acids: L ( S)

N

H

O

O

Bend in backbone introduced by proline

SS

NH

NH

NH

NH

NH

O

O

R

R

O

O R

R

Distant regions brought into juxtaposition by disulfide bond

HORSERADISH PEROXIDASE C chain a

α-helix

β-sheet

Cys 11-Cys91

N

N

NH2

OO

HOOH

H N

N

O

H2 N N

N

O

H OOH

HO PO

O -O ][[ ] HO P

O

O -O ][[ ]

d e o xyg ua no s ine , d G uo

[g ua n y lic a c id ]

N

N

NH2

N

N

O

H OOHHO P

O

O -O ][[ ]

HN

N

O

O

C H3

O

HOOH

HO PO

O-

O ][[ ]

d e o xyc yt id ine , d C yd

[d e o xyc yt id y lic a c id ]

g ua n ine , G ua {o r G} c yto s ine , C

a d e n ine , A d e {o r A

d e o xya d e no s ine , d A d o

[d e o xya d e n y lic a c id ]

} th ym ine , T

d e o xyth ym id ine a c id , d T h yd

[d e o xyth ym id y lic a c id ]

n u c le o b a s e d e o xy n u c le o s id e d e o xy n u c le o tid e(n u c le ic a c id )

b a se b a se + d e o x y r ib o se b a se + d e o xy r ib o se -5 '-p ho s-p ha te

1

23

6 7

89

1'

2'3'

4'5'

12

3

4

5

6

1'

2'

3'

4'

5'

n um b e r ing c o n ve nt io n

p u r in e s p yr im id in e s

phosphodiester

{ bond

3’

53’

5’

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.

Hoogsteen pairing

major groove

minor groove

B-DNA

Z-DNA

H-bonding edge

antisyn

Orientation of base around glycosydic linkage

Hoogsteen-like pairing with modified dGuo in syn orientation

N

N

NN

O

H2N H

HO

NH2

O NN

NHN

A T C A G A

T A G T C T

5' 3'

3' 5'

B

PP

B

P

B

P

B

OH

Common conventional representations of DNA

forward

backwardA + B A-B + H2O

EQUATIONS FOR THERMODYNAMICS H ≡ enthalpyE ≡ internal energyP ≡ pressureV ≡ volumeChange in enthalpy: ΔH = ΔE + P ΔV S ≡ entropy Change in free energy: ΔG = ΔH – TΔS For the reaction as written:  ΔG < 0, spontaneousΔG > 0, not spontaneous- work must be put into the system to drive it in the forward directionΔG = 0, the system is in equilibrium K ≡ equilibrium constant, ratio of concentrations of products to reactants:  

ΔG = ΔGo + RTln KR ≡ gas constant (= 1.98 cal/mole-oK = 0.00198 kcal/mole-oK)T in oKΔGo = ΣGo

products - 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

]][[

]][[ 2

BA

OHBAK

forward

backwardA + B A-B + H2O

 ΔG = -RT ln K ΔG = +6 kcal/moleR = 0.00198 kcal/mole-oKT = (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) = -10.2K = 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-dN1 x 10–3 MVirtually 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 

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?

Q: What is the equilibrium concentration of dinucleotide from a 1 x 10-3 M initial concentration?

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

 

N

N

N

N

O

OHOH

O

NH2

P

O

O-

OP

O

O-

OP

O

O-

O-

O-

P

O

O

5’-dNMP-3'-O

5'-dN'

P

O-

O-

O 5'-dN

5’-dNMP-3'-O OH

-OH

5'-dNMP + 5'-dN'MP

transition state

Hydrolysis of phosphodiester linkage

products

reactants

reaction coordinate

G

G‡

G

transition state

G

reaction coordinate

reactants

products

ΔG‡

ΔG

Effect of enzyme on ΔG‡

THREE STAGES OF REPLICATIONinitiation – recognition of originelongation – extension by replisometermination

2 pi

+OH

B'

P3

proofreading

OH

B'

P3+ ?

B

P P

B

P

B B

OHP

P3

B'

OH+

B

P P

B

P

B B

PP

B'

OH

proofreading B

P P

B

P

B B

OHP +

B'

OHP

5’ 3’ addition

3’ 5’ addition

+

P-P

H2O2Pi

P P

B B

P

B B P3 P

BP P

B' B

P

B BP3

P P

B B

P

B B P

B’'

OHP3

OH

B‘’

P3

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 extenda 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.

pol α, 5'3' synthesis but no 3'5' exonuclease capabilitypol β, 5'3' synthesis with no 3'5' exonuclease capabilitypol δ, 5'3' synthesis + 3'5' exonuclease capabilitypol ε, 5'3' synthesis + 3'5' exonuclease capabilitypol γ, 5'3' synthesis + 3'5' exonuclease capabilitypol 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

PROKARYOTIC POLYMERASES

EUKARYOTIC POLYMERASES

3'-OH

5' 3'

Direction of replication fork progression

SSBs

1

2 3

4

τ

β-clamp

DNA pol α RNA priming + short 3 – 4 base DNA extension (iDNA; i = initiation)

DNA pol δ Strand extensionPCNA (proliferating cell nuclear antigen) Processivity (equivalent function to β-clamp)RFC (replication factor C) Loads pol δ and PCNA at end of iDNAFEN1 and Dna2 (5 3 exonuclease) Removal of RNA primerDNA ligase I Seal nicksRPA (replication protein A) Single strand binding proteinsMCM (minichromosome maintenance) Helicase function

Some Eukaryotic Replication Proteins

MODEL OF EUKARYOTIC REPLICATION FORK

                                                                                                                                                   

prokaryotic origin of replication

G (*A) T C

C T (*A) G

G (*A) T C

C T (A) G

control of replication at prokaryotic origins

parent duplex parent + daughter duplex

*A =

fully methylated hemi-methylated

N

NNH

N

HN

CH3

N6-MeAde

% of origin function

Autonomously replicating sequence: ARS

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.

Model illustrating how Cdt1 and geminin limit DNA replication to exactly one round per cell cycle

Double stranded DNA

template DNA: antisense/anticoding strand

DNA not copied: sense/coding strand

DNA-RNA hybrid

mRNA coding strand

template DNA: antisense/anticoding strand

Codons are represented as the mRNA coding strand.

OB

OH

HO

DNA RNA

HN

NH

O

O

CH3HN

NH

O

O

thymine uracil

deoxyribose ribose

OB

OHOH

HO2'

DNA-RNA distinctions

U C A GU UUU

UUCUUAUUG

UCUUCCUCAUCG

UAUUACUAAUAG

UGUUGCUGAUGG

C CUUCUCCUACUG

CCUCCCCCACCG

CAUCACCAACAG

CGUCGCCGACGG

A AUUAUCAUAAUG

ACUACCACAACG

AAUAACAAAAAG

AGUAGCAGAAGG

G GUUGUCGUAGUG

GCUGCCGCAGCG

GAUGACGAAGAG

GGUGGCGGAGGG

Phe

Leu

Leu

Ile

Met

Val

Ser

Ser

Ala

Pro

Thr

Tyr

STOP

His

Gln

Asn

Lys

Asp

Glu

Cys

Trp

Arg

Arg

Gly

STOP

5'NNN3'

anticodon

D arm

anticodon arm

TC arm

Amino acid

dihydrouridine D

HN CH2

CH2N

O

O

pseudouridine

HN NH

C

O

O

Yeast phe tRNA

(not charged with aa)

3-terminus

5-terminus

stick ribbon

5AGC3

3UCG5

AGC

GCU

1 2 3

codon

anticodon

codon

anticodon

123

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

Wobble hypothesis: rules for codon/anticodon pairing

Genes VIII, Fig. 6.2

protein synthesis

PROKARYOTIC mRNA/PROTEIN SYNTHESIS EUKARYOTIC mRNA PROCESSING

Genes VIII, Fig. 5.13

Genes VIII, Fig. 5.17

5-CAPPING OF EUKARYOTIC mRNA

splice

exon

introns

exonexon

G

C

U

C

A

C

G

A

G

U

NN

NN

N

NN

NN

N

G C U C A N N N N N N N N N N U G A G C

STEM LOOP

Subunit (molecular weight) Function

2 x (40 kD) enzyme assembly, promoter recognition

(155 kD) catalytic center

(160 kD) catalytic center

(32-90 kD) promoter specificity

ω(10 kD) enzyme assembly

Subunits of prokaryotic RNA polymerase

Cat

alyt

ic c

ore

2′ω= holoenzyme

-10 consensus sequence T80 A95 T45 A60 A50 T96

-35 consensus sequence T82 T84G78A65C54A45

coding strand

start point

+1

5' 3'

upstream, -n downstream, +n

2-D REPRESENTATION OF E. coli RNA polymerase

–10 consensus sequencedownstream – direction of transcription

NTP access channel

-1

-17

coding

anticoding

active site

N-terminalthumb

fingers

palm

“Hand” convention for representation of catalytic unit of polymerases

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 down-stream.

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)

CH2

S

OH

HOHO

OH

O

truncation at hinge

truncation at hinge

truncation at point of hinge attachment

Tetramer, with two of the tetrameric units selected

B. Rotated 90o around core axis

Headpiece (hinge + HTH motif)

A. Looking down DNA helix

Lac repressor dimer bound to operator

anti-inducer

o-nitrophenylfructose

(ONPF)

hinge

Lac repressor + IPTG truncated at hinge.

Lac repressor + ONPF truncated at oligomerization domain

Contrast inducer-bound and active lac repressor

a b

Structure and binding of tetrameric lac repressor protein

OO

OOH

P

O

N

N

NH2

N

N

O

cyclic AMP (cAMP)