Glutamate mutase glutamate NH2 mutase NH2
HO2C HO2C CO2H CO2H Coenzyme B12 CH3 L-glutamate (2S,3S)-3-methyl-L-aspartate
methylaspartate
Coenzyme B12-Co
adenosyl
His-16
pdb code: 1I9C
241
Nucleic Acids 1869: Miescher- gelatinous material from cell nuceli of white blood cells containing organophosphorous compounds- nuclein (chromatin)- discovery of nucleic acids 1891: Kossel- identified the DNA bases A, T, G and C identified D-ribose in nucleic acids 1889: Altman- purified DNA 1901: Ascoli- identified U in RNA 1910 : DNA and RNA realized to be separate entities. DNA (thymus) RNA (yeast) 1929: Levene & Jacobs- identified 2’-deoxyribose in DNA 1920’s - 1950’s: structures of nucleosides and nucleotides - Alexander Todd (Nobel Prize, 1959) 1928: Griffith- first to propose that DNA was genetic material; not widely accepted G 1931: Levine & Bass- first proposed structure. Believed to be part O O O P O C of chromosome physiology, but NOT genetic material O 1944: Avery, MacLeod & McCarty- Strong evidence that DNA O O O P O is genetic material O O O P O 1950: Chargaff- careful analysis of DNA from a wide variety of O O O T O P O organisms. Content of A,T, C & G varied widely according O to the organism, however: A=T and C=G (Chargaff’s Rule) O A 1952: Hershey & Chase: followed the transfer of 32P-labelled DNA from T2 bacteriophage to infected bacterial cytoplasm (1969 Nobel Prize) 1953: Watson & Crick- structure of DNA (Nobel Prize with M. Wilkens, 1962)
242
121 Nucleoside, nucleotides and nucleic acids Blackburn et al. Ch. 2
base base
phosphate sugar sugar
nucleoside nucleotide nucleic acid
243
Sugar: D-ribose
CHO HO OH H OH HO H O O H H H OH H H H H H OH H OH HO OH OH CH2OH HO β-D-ribofuranose α-D-ribofuranose
HO OH O H H H H HO H
β-D-2-ribofuranose
Stereochemistry: As drawn, the side above the plane of the ring is β the side below the plane of the ring is α 244
122 Nucleosides vs Nucleotides
O HO O B HO P O O B O
HO X HO X
ribonucleoside (X=OH) ribonucleotide (X=OH) deoxyribonucleoside (X=H deoxyribonucleotide (X=H
O O O O O HO P O P O O B HO P O P O P O O B O O B O O O O O O P HO X HO X O O OH
nucleotidediphosphate nucleotidetriphosphate 3',5'-cyclic phosphosphate
245
NH O O O N 2 O O N NH2 -O P O P O P O H2O -O P O P O O N N O N N - - O- - - 2- O O N O O N + PO4H HO OH HO OH ATP ADP
+ ~32 KJ/mol (7.3 Kcal/mol)
Hydrolysis of ATP is used to drive many biochemical reactions
Phosphoryl transfer reactions:
NH OH O O O N 2 HO -O P O P O P O O N N HO O - - - O O O N OH OH HO OH
- N NH2 -O O O O 2- OPO3 O P O P O O P O N N HO - - - + ADP HO O O O N HO O HO O HO OH OH OH OH OH 246
123 Bases A. Purines O NH2 O
N N N N HN HN
N N N N H2N N N R R R Adenine (R=H) Guanine (R=H) Hypoxanthine (R=H) Adenosine (R= furanose, A) Guanosine (R= furanose, G) Inosine (R=furanose, I)
B. Pyrimidines
NH2 O O NH2
N NH NH N
N O N O N O N O
R R R R Cytosine (R=H) Thymine (R=H) Uracil (R=H) 5-Methylcytidine Cytidine (R=furanose, C) Thymidine (R=furanose, T) Uridine (R=furnaose, U) (R=furnaose)
DNA contains A, C, G, T all with 2'-deoxyribose! RNA contains A, C, G, U all with ribose! ! The stereochemistry of the base is β 247
Numbering System
4 O O6 O O 5' 7 4 7 6 3 HO O Base N 5 5 5 N 5 1 NH 2 N 3 4 NH N 6 1 N 4' 1' 8 8 2 2' 6 1 2 N 6 2 N O O2 N N 9 4 N 2 NH2 N HO 3' OH 9 H 4 3 R 3 1 R Ribonucleoside Purine Guanosine Pyrimidine Uridine
248
124 Nucleoside Conformation:
O O O O N N NH NH HN HN N N N NH2 N O H2N N O N HO HO HO HO O O O O
OH OH OH OH Anti conformation Syn conformation 5-member ring conformation: envelope
N O 5.9 Å R RO H O N O O N RO NH O N H H N H NH O H N 7.0 Å NH2 H R NH2
C2' - endo conformation! C3' - endo conformation! found in B-form DNA! found in A-form DNA! 249
Watson & Crick: double helix Initial “like-with-like”, parallel helix: Does not fit with with Chargaff’s Rule: A = T G = C
H H N N N dR dR dR dR N O N N N N O N H N O N N N N H H H O O H H H N N H H H H N N N O N N H N O N N O N N N dR dR dR dR N N N H H purine - purine pyrimidine - pyrimidine
Wrong tautomers !!
Watson, J. D. The Double Helix, 1968 250
125 Complimentary Base- Pairing in Nucleic Acids: antiparallel double helix
Antiparallel C-G Pair Antiparallel T-A Pair H H O H N N N H O N 5' 5' N H N N RO N H N N RO 5' N N O 5' RO N N O O RO O O O H N OR H OR 3' 3' OR OR 3' 3'
H 6 Hydrogen Bond H N Hydrogen Bond 4 Hydrogen Bond Hydrogen Bond 4 Donor N O6 O O H N N Donor N H O N A cceptor Acceptor N1 Hydrogen Bond Hydrogen Bond Hydrogen Bond N N N3 N Hydrogen Bond N3 N H Acceptor Acceptor N H N N1 Donor Donor N N R N N R R O Hydrogen Bond 2 R O H N Hydrogen Bond Acceptor O N2 H Donor
“Molecular Structure of Nucleic Acids” Watson J. D.; Crick, F. H. C. Nature 1953, 171, 737-8
251
B-DNA
Minor Minor groove groove
Major Major groove groove
pdb code: 1bna 252
126 DNA Grooves
major groove
cytidine guanosine
minor groove
major groove
thymidine adenosine
minor groove
253
Writing DNA sequences DNA Sequences are written 5’ to 3’
5’-ATCGCAT-3’
5’-d(ATCGCAT)-3’
A T C G C A T 5' 3' HO P P P P P P OH
254
127 Melting Temperature (Tm): a measure a duplex stability Double Δ Single stranded strand Hybridzed: Unhybridized: highly ordered less ordered, structure random coil
Base stacking causes a deceases in the net UV absorbance when DNA is hybridized (double stranded) versus unhybridized.
Upon thermal denaturation (melting), the UV absorbance increases: hyperchromicity
Blackburn et al. Ch. 2.5.1, 11.1.1 255
DNA melting curve: 5’-d(GCT AGC GAG TCC)-3’! 0.2 3’-d(CGA TCG CTC AGG)-5’ annealing single )
m stranded n melting 0.15 0 6 2 ( 0.1 s b A 0.05 double stranded Tm= 65 °C 0 0 20 40 60 80 100 T (°C)
Tm is often taken as a measure of DNA duplex stability
Tm is a measure of ΔG not ΔH
The Tm is dependent upon the length and sequence of the oligonucleotide (CG/AT ratio) and the ionic strength of the medium 256
128 DNA processing enzymes: Blackburn et al. Ch. 3.6, 5.3, 6.6.4 DNA replication: Helicase: Unwinds double stranded DNA DNA polymerase: replicates DNA using each strand as a template for the newly synthesized strand.
“It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” 257 Watson & Crick
DNA Polymerase: the new strand is replicated from 5’ to 3’ DNA polymerase are Mg2+ ion dependent Details regarding the mechanism of recognition of dNTP incorporation for the growing DNA strand are not fully understood
258
129 DNA Ligase: ATP-dependent enzyme that will join (ligate) two DNA segment by catalyzing the formation of the phosphodiester bond of a terminal 5’-phosphate of one oligonucleotide and the 3’-hydroxyl group of another oligonucleotide.
5' 3' 5' 3' 2- 2- O3PO OH O3PO OH
2- OH OPO3 3' 5'
5' 3' 5' 3' 2- 2- O3PO OH O3PO OH 5' 3' 2- O3PO OH
OHOPO 2- DNA Ligase 3 Phosphodiester 3' 5' bonds
DNA Ligase
259
Restriction Enzymes (endonucleases): enzymes that will cleave double stranded DNA at specific, known sequences.
5’-d(G-A-A-T-T-C)-3’! EcoR I 3’-d(C-T-T-A-A-G)-5’! ! ! 5’-d(G-G-A-T-C-C)-3’! 3’-d(C-C-T-A-G-G)-5’! BAM HI
5' 3'
3' 5'
restriction enzyme
2- OH O3PO 5' 3' “sticky ends”
3' 5' 2- O3PO OH
Werner Arber, Daniel Nathans and Hamilton Smith 260 1978 Nobel Prize in Medicine & Physiology
130 Type I: cleaves at a site very distant from the recognition sequence; Mg2+, SAM and ATP dependent. Type II: cleaves DNA at or near the recognition sequence; Mg2+ dependent. i.e., EcoR I, BamHI, Bbs I
BbsI: GAAGACNNNNNNN! !CTTCTGNNNNNNN Recognition sequence Cleavage: blunt end
5' 3'
3' 5'
Type 2 restriction enzyme
2- OH O3PO 5' 3'
3' 5' 261 2- O3PO OH
Polymerase Chain Reaction (PCR): method of amplifying DNA using DNA polymerase and cycling temperature
Heat stable DNA Polymerases: Taq: thermophilic bacteria (hot springs)- no proof reading Pfu: geothermic vent bacteria- proof reading
Mg 2+ two Primer DNA strands (synthetic, large excess) one sense primer and one antisense primer one Template DNA strand O O O -O P O P O P O O B dNTP’s O- O- O-
HO
KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction (PCR) method.
Blackburn et al. Ch. 5.2.3, 5.4.2 262
131 A typical PCR temperature cycle
Denaturation: 94 °C 0.5 - 1 min
Annealing: 55-68 °C 0.5 - 1 min 5 °C below the
Tm of the primer
Extension: 72 °C 1 min + 1 min per Kb of DNA
# of cycles 25 - 35
Final extension 72 °C 10 min
263
Polymerase Chain Reaction 5' 3'
5' 3' 95 °C
denaturation 3' 5'
3' 5'
5' 3' 72 °C 55 - 68 °C Taq, Mg 2+, dNTPs 3' anneal 3' extension (+) and (-) primers 3' 5'
5' 3'
5' 3'
3' 5' 95 °C 3' 5' 5' 3' denaturation 5' 3' 2nd cycle 3' 5'
2 copies of DNA 3' 5'
amplification of DNA
264 For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html
132 $$ The Power of Compounded Interest $$
1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2 = 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2 = 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576
In principle, over one million copies per original, can be obtained after just twenty cycles 265
Oligonucleotide-Based Site-directed mutagenesis
DNA mRNA protein
Blackburn et al. Ch. 5.6 266
133 3’-GAG ATG ACA CCC AAA-5’! 5’-CTC TAC TGT GGG TTT-3’! ! -Leu Tyr Cys Gly Phe-! ! ! ! 3’-GAG ATG CGA CCC AAA-5’! 5’-CTC TAC GCT GGG TTT-3’! ! -Leu Tyr Ala Gly Phe-!
267
Restriction Map
23 BspD I 23 Cla I EcoR I 4361 29 HinD III Aat II 4286 185 EcoR V Ssp I 4170 229 Nhe I 375 BamH I
Sca I 3846 562 Sph I 622 EcoN I Pvu I 3735 651 Sal I Pst I 3609 Ase I 3539
Bsa I 3435 939 Eag I pBR322 972 Nru I Eam1105 I 3363 1063 BspM I
4363 base pairs Unique Sites
1353 Bsm I 1369 Sty I AlwN I 2886 1425 Ava I 1444 Bal I 1444 Msc I 1650 Bsg I 1664 BspE I 1664 BspM II Afl III 2475 1668 BsaB I Nde I 2297 2066 Pvu II Xca I 2246 2124 Esp3 I Bst1107 I 2246 Bsa I 2227 Tth111 I 2219
268
134 EcoR I EcoR I EcoR I Cys Ala BamH I
Cys Ala EcoR 1 BamH1 BamH I BamH I
insert
envelope
BAM H1 Cys plasmid (template) 3'-GAT CCC NNN CTC TAC ACA GGG TTT NNN------5' (−) antisense 5'-CTA GGG NNN GAG ATG GCT CCC AAA NNN-3' (+) sense Ala PCR primer with mutation
PCR amplifies a new insert with the mutation
Cut the PCR fragments with EcoR1 and BamH1
Ligate the new insert (with the desired mutation) back into the envelope
MICHAEL SMITH, 1993 Nobel Prize in chemistry for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. 269
Amplification of the mutant sequence by PCR ACA Codes for Cys TGT
Denature and anneal primers (-) Primer contains the mutation ACA for Ala GCT TGT (+) primer
extend
ACA ACA GCT TGT
Denature and anneal primers
ACA ACA GCT GCT
GCT TGT
extend
ACA ACA GCT GCT
CGA ACA GCT TGT 270
135 ACA ACA GCT GCT
CGA ACA GCT TGT
Denature and anneal primers
ACA ACA GCT GCT
GCT GCT
CGA ACA GCT GCT
GCT TGT
Extend
CGA ACA GCT GCT CGA CGA GCT GCT
CGA ACA GCT GCT
CGA ACA GCT TGT DNA strands with the desired mutation are being amplified much faster than strands without the mutation; after ~25 rounds of PCR, the amount of DNA that does not have the desire mutation become 271 negligible (<< 1 in a million)
Nucleoside Synthesis: Blackburn et al. Ch. 3.1 important class of chemotherapeutic agents (anticancer and antivirals) important reagents for biotechnology Anti-Cancer Nucleosides
F O F3C O NH2
NH NH N N N HO N HO O HO O O O O O F HO HO HO F
Anti-Viral Nucleosides
O NH NH2 2 H2N O N NH N N N NH HO N N HO N HO N N OH O HO O O O O O O N O O S
N3 AZT ddC ddI d4C (-)-3TC
272
136 Chemical Synthesis of Ribonucleosides via Vorbrüggen Glycosylation
SiMe3 O Cl - OTMS N N + AcO N OTMS AcO O O OAc N OTMS AcO O O O SnCl4, CH3CN AcO OAc AcO O + AcO O
O O
NH NH HO N AcO O N NH3, MeOH O O O
AcO OAc HO OH β-Ribonucleoside
273
The stereochemistry of the C2-ester group of the furanose-tetraester controls the stereochemistry of the glycosylation reaction
O OTMS N NH AcO O AcO N OAc N OTMS O O
SnCl4, CH3CN AcO OAc AcO OAc
ribose-tetraacetate β-1'-stereochemistry
O OTMS N NH AcO O AcO N OAc N OTMS O O
SnCl4, CH3CN AcO OAc AcO OAc
arabinose-tetraacetate α-1'-stereochemistry
274
137 Exocyclic amino groups of C, A and G require protecting for the Vorbrüggen Glycosylation reaction
O OTMS R R HN N
O N R= H, CH TMSO N H 3
O OTMS
HN R N R
N N
O N TMSO N H R= CH3, CH(CH3)2
O OTMS HN Ph N Ph N N N N N N H N N (H3C)3Si
OTMS O N N N OTMS NH O N N N R N N N R H H (H3C)3Si R= CH3, Ph 275
Chemical Conversion of Ribonucleoside to 2’-Deoxyribonucleoside: Free radical deoxygenation of the 2’-hydroxyl group
O O
NH (iPr2SiCl)2O, NH PhOC(S)Cl, HO N pyridine O iPr O N pyridine O O Si iPr O O HO OH Si O OH β-Ribonucleoside iPr iPr
O O
NH NH 1) nBu3SnH, AIBN iPr O N O PhCH3, reflux HO O N Si O O iPr + - O 2) nBu4N F , THF Si O O OPh HO iPr iPr S β−2'-Deoxyribonucleoside
276
138 Solid-Phase Oligonucleotide Synthesis!
S S S OH O O O O I I I L L L OH O Si (CH2)3 NH2 O Si (CH2)3 N C (CH2)2 C OH I I I H C OH C O C O A A A
DMTrO B1 O DMTrO B1
O HO B O N (iPr)2 S 1 P O O I O O L Cl3CCOOH O Si (CH2)3 N C (CH2)2 C O CN I H (detritylation) C O O O N NH 1H-tetrazole A (coupling) N N
HO DMTrO B2 B1 DMTrO B2 O O O
1) Ac2O (capping) Cl CCOOH O O O O 2) I2, H2O (P oxidation) O O 3 CN P CN CN P O P O Repeat Cycle O O B1 B O B1 O O O
O O O
HO B2 O NH , H O Cl3CCOOH 3 2 O O deprotection P O - -and- O cleave from B1 solid support O 277
Blackburn et al. Ch. 4 HO
The Protecting Groups for Solid Phase DNA Synthesis: 5’- hydroxyl group
R1 Trityl R1= R2= R3 = H Tr-OR (p-Methoxyphenyl)diphenylmethyl ether R1= R2= H, R3 = OCH3 4'-methoxytrityl MMTr-OR R2 C O R Di-(p-methoxyphenyl)phenylmethyl ether R1= H, R2=R3 = OCH3 4',4'-dimethoxytrity DMTr-OR
Tri-(p-methoxyphenyl)methyl ether R1= R2= R3 = OCH3 R3 4',4',4'-trimethoxytrityl TMTr-OR
Relative rate of hydrolysis: O O
HN HN 80% AcOH R O O N (aq) HO O N O 20°C O
HO OH HO OH R = Tr 48 hr. R= MMTr 2 hr. R= DMTr 15 min.! R= TMTr 1 min. (too labile to be useful) 278
139 Protecting groups for the exocyclic amino groups
O O
HN HN Ph O O N N N N N NH O NH N O N N N N N N N N N H dR dR dR dR
O O OPh HN HN O N N N N NH O OPh O N N N N N N H dR dR dR
All are removed with NH3/H2O at 80° C
279
Phosphoramidite reagents: the monomeric building blocks for solid-phase DNA synthesis
N O HO B DMTrO B P CN DMTrO B O DMTr-Cl O O Cl pyridine [(CH3)2CH]2NH HO HO O NC P Base is protected O N[CH(CH3)2]
for solid-phase RNA synthesis
HO B DMTrO B Si Cl DMTrO O B DMTrO O B O DMTr-Cl O + pyridine Ag+ HO OH HO OH HO O Si Si O OH
Base is protected 70 : 30
N O [(CH3)2CH]2NH P CN Cl
DMTrO O B silyl group removed with fluoride ion (F-)
O O Si NC P O N[CH(CH3)2] 280
140 DNA Sequencing: Maxam-Gilbert Sequencing Blackburn et al. Ch. 5.1 sequence selective chemical cleavage Detection: 5'-32P-labeling
A: Cleavage at the 3'-side of guanosines with a) dimethylsulfate b) Δ, c) piperidine.
32 O 32 O 32 P P H3C P N NH N NH O + O O N N NH H CO S OCH O O N N NH O 2 3 3 2 O OH O H2O, Δ
O O O O O O H3C O - P - P N P O O O O lose NH -O O N N NH2
32 P 32P 32P
O OH H O OH H O OH H O N + N +
O O H H H O O O- O P - P H -O O O O N P -O O
281 Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499-558
Maxam-Gilbert Sequencing
B. Cleavage at the 3'-side of adenosines and guanosines with a) dimethyl sulfate
b) HCO2H, c) piperidine.
32 NH2 NH2 P 32P 32P N N N N O + O N N H CO S O N N O O 3 OCH3 O O OH HCO2H, H2O O CH3
NH2 O O O O O O P N N P - - P - O O O O lose + O O N H N
CH3
32P 32P 32P
O OH H O OH H O OH H O N + N +
O H H O O H O - P P H O - -O O N O O O P -O O
282
141 Maxam-Gilbert Sequencing
C. Cleavage at the 3'-side of thymidines and cytidines with a) hydrazine, b) Δ, c) piperidine.
+ 32P 32P H O 32P O NH HN 2 H HN O N O CH3 NH O CH O N 3 O N O H2N NH2 O O O N O HN NH2 O H2O O O O O P O P -O O P -O O -O O
32P 32P
O O OH NH O 2 O OH NH2 N N
O H H H O O- P H O -O O N P -O O
283
Maxam-Gilbert Sequencing
D. Cleavage at the 3'-side of cytidines with a) hydrazine, 1.5 M NaCl, b) Δ, c) piperidine.
32 NH P + 2 32 H+ H P NH2 HN 32P NH NH 2 N O N O O O N NH O O N H N NH O O O N 2 2 HN NH2 O 1.5 M NaCl O O P O O - O P O O P -O O -O O
32P 32P 32P NH O O 2 H O O OH NH O OH NH N NH 2 2 O O N N N
NH2 + O H H O H O- O P H O O -O O N P P -O O -O O
284
142 Maxam-Gilbert Sequencing Separation and detection of 32P-labeled DNA fragments by polyacrylamide gel electrophosesis (PAGE). DNA fragments separated based upon charge, which is proportional to the length of the DNA fragment.
G A & G C C & T Larger 3’ fragments G T A A C G T A A T C A C Smaller A fragments G 5’-32P
285
Sanger Sequencing Enzymatic replication of the DNA fragment to be sequnced with DNA polymerase, Mg+2, ddNTP's and 32P-labeled primers.
ddNTP’s
NH2 O
N N NH O O O O O O N N N O -O P O P O P O -O P O P O P O O O O- O- O- O- O- O-
ddATP ddTTP
O NH2
N NH N
O O O N O O O N O N NH2 -O P O P O P O O -O P O P O P O O O- O- O- O- O- O-
ddGTP ddCTP
286
143 Sanger Sequencing ddA ddG ddC ddT 5' Larger C G fragments A T T A T A G C C G A T T A T A A T G C T A G C Smaller T A fragments C G 3'
5' 3' 32P 32P-5' 3' primer template
287
Sanger sequencing with flourescent ddNTP's
H H O N O NH2 N O
HN H C N H C 3 N 3 N O N O N -3 O -3 O H O P O H3O9P3O 3 9 3 (CH2)2 (CH2)2 O O H3C CH3
- O O O - O O O CH CH3 CH3 CH3 3 ddT ddC
O O O O N N CH 3 HN CH3 HN
H C CH 3 3 NH2 O
- N NH O O O O O O - N N N N NH -3 H O P O -3 2 3 9 3 H3O9P3O O O
ddA ddG
Excitation: ~ 490 nM, Emission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm
288
144 Sanger sequencing using flourescent ddNTP's terminated DNA strands are separated by capillary electrophoresis
289
Pyrosequencing 5'-ACCTTGATACCATCTAGGA------3' AGATCCT------5'
dNTPs are added dTTP, Mg2+, sequentially one at a time DNA polymerase
5'-ACCTTGATACCATCTAGGA------3'- TAGATCCT------5' + Three enzyme system where the O O O incorporation of a complimentary O P O P O O S O P O 3 O A O O O Adenosine- nucleotide opposite a template DNA 5’-phosphosulfate strand results in the emission of a HO OH (APS) photon. ATP-Sulfurylase
O O S 2- O P O P O P O ATP + SO4 O A O O O
HO OH Luciferase, O2 is used in place of ATP - not a substrate for ATP-sulfurylase oxyluciferase + P O 2- + AMP + photon & luciferase 2 7
Ronaghi, M., Genome Res. 2001, 11, 3-11 290
145 5'-ACCTTGATACCATCTAGGA------3' TAGATCCT------5'
dNTPs are added dGTP, Mg2+, sequentially one at a time DNA polymerase
5'-ACCTTGATACCATCTAGGA------3'- GGTAGATCCT------5' +
O O O 2 O P O P O O S O P O 3 O A O O O
HO OH ATP-Sulfurylase
2- 2 ATP + SO4
Luciferase, O2
2- oxyluciferase + P2O7 + AMP + 2 photon
291
pyrogram
5’-----TCCCCACCGAAACCCCAACGTCAAC------3’ AGGGGTGGCTTTGGGGTTGCAGTTG------5’
292
146 Apyrase is a fourth enzyme that is added that will hydrolyze the dNTP and ATP to the corresponding dNMP and PPi between dNTP additions. The rate of hydrolysis of apyrase is slower that than the other three enzymes.
Alternatively, a biotinated template is used which is bound to streptavidin coated beads. The immobilized template DNA is washed between dNTP addition
streptavidin•biotin--–––-5’-ACCTTGATACCATCTAGGA------3’ -15 K D= 10 AGATCCT------5'
O O
HN NH HN NH H O-DMTr H H H H N i S CO2H S N Pr2 O P CN Biotin O O
293
Sequencing by Reversible Chain Termination N3 Dye – O2C PPPO O B linker O CO – P 2 O N 3 – CO2 tris(2-carboxyethyl)phosphine (TCEP): 3’-O-blocked fluorescently Reduces azides to an amino groups labeled dNTP read fluorscence = a specific base
N read fluorscence 3 = a specific base Dye linker O linker OH linker OH N3 DNA polymerase 3' O TCEP N 3' OH linker O 3 DNA polymerase 3'-O-blocked- O Dye fluorescent dNTPs 3' Etc. 3'-O-blocked- N fluorescent dNTPs 3
chain elongation is blocked
chain elongation is blocked
C T C T G C A G A G C C C C C C
147 Reversible Chain-Terminating, Fluorescent dNTPs
N N O N O B 3 H F O O O N F N O N N O N H H HN O
– O N CO2 O N3 O NH H O 2 O O N N N O N H H O PPPO O N N 3 N dTTP (blue) O PPPO O N3 H H N O N Et + Et dATP (yellow)
CO – O N O 2 3 H NH O O N 2 N O N H H – N O SO3 O N
O N PPPO O N3 O N O 3 H O O O N HN N O N H H O – H2N SO3 dCTP (green) N N N
O
PPPO O N3 dGTP (red)
Light-Directed, Spatially Addressable Parallel Synthesis
hν
OP OP P P P P P P P P P P A A P P P P OH OH O O O O O O O O O O Coupling O O O O O O
A-OP
hν
OP OP OP OP OP OP Coupling A A P P P P A A B B P P O O O O O O O O O O O O B-OP
hν
OP OP D D OP OP OP OP OP OP P P P P O O P P P P Coupling A A B B C C A A B B C C A A B B C C O O O O O O O O O O O O O O O O O O D-OP
296
148 Photoremovable protecting group: o-nitrobenzyl ether derivatives
O
O O O B Mechanism: hν (λ ~ 350 nm) NO2 O NC O R P O R O N[CH(CH3)2] O hν H O H O O N O O N O O
O R O R O R O O H O O + O O N O N + O H O N O O + OH H-OH2
O R O O O O ROH + O N O N O H O
H2O 297
Spatially addressable: 8-mer chip: 65, 536 different sequences 12-mer chip: 1,677,216 different sequences DNA fragment DNA–fluorophore Place DNA on the chip, then wash away non-specific hybridization after 1-10 hrs. Raise temperature and “melt” away partially hybridized sequences.
3’-ACGGTGCG! CGGTGCGA ! !GGTGCGAG! GTGCGAGA! TGCGAGAA! GCGAGAAT etc! ! 3’-ACGGTGCGAGAAT---5’ (from the chip)! 5’-TGCCACGCTCTTA---3’! 298
149 Nucleotide Biosynthesis
CHO ATP AMP H OH PO PO O OH O H OH OPP H OH HO OH HO OH CH3OP 5-Phosphoribosyl- D-ribose-5-phosphate 1- pyrophosphate (PRPP)
Blackburn et al. Ch. 3.4, 3.5 299
Purine Biosynthesis " O " H N glutamine glutamate 2 H X PO O NH2 glycine PO HN 10-formyl- + ATP O PRPP O tetrahydrofolate amidophospho- glycinamide GAR ribosyl transferase HO OH ribonucleotide (GAR) synthase HO OH transformylase O O glutamine glutamate + N NH + ATP ADP+ Pi NH ATP H H ATP PO N PO HN - H2O O NH2 O PO O HN O NH formylglycinamidine 2 Aminoimidazole + ribonucleotide (FGAM) synthase (AIR) synthase HO OH HO OH HO OH - O CO2 - - N CO2 CO2 - aspartate, N HCO3 , ATP ATP N H -fumaric acid No biotin PO O N NH -ADP, -Pi 2 aminoimidazole PO O N NH AIR succinylocarboxamide 2 carboxylase adenylosuccinate ribonucleotide lyase HO OH (SAICAR) synthase HO OH
O O O N NH 2 N N 10-formyl- NH2 NH - H2O PO N tetrahydrofolate O NH CHO PO N 2 PO O N N IMP O N aminoimidazole H cyclohydrolase carboxamide HO OH ribonucleotide (AICAR) transformylase HO OH HO OH Inosine monophosphate (IMP) 300
150 Folate derivatives – one-carbon donors
- O CO2 - O CO - O CO2 N 2 H O N HN N - O CH3 N H CO2 H H N N N - HN N CO - HN N CO2 2 N H H N N N H2N N N H O H2N N O 2 H H H 10-formyl-tetrahydrofolate 5,10-methylene-tetrahydrofolate 5-methyl-tetrahydrofolate
" O " O = = " -or- " " " X CH2OH = X CH3 H X H H
- O CO - O CO2 2 dihydrofolate O N O N reductase H H N N - HN N CO - HN N CO2 NADH 2 H H H N N N H2N N N 2 H Folic Acid Dihydrofolate - O CO2 - O CO2 NH2 N H dihydrofolate N O N - reductase H H N N CO2 N HN N CO - R NADH 2 H N N N H 2 R= CH3, methotrexate H2N N N R= H, aminopterin H Tetrahydrofolate Dihydrofolate reductase inhibitors 301
5,10-methene-THF to 5-formyl-THF - O CO2 O CO - 2 5,10-methylene-THF 5,10-methenyl-THF N cyclohydrolase N dehydrogenase H H H O NADP HN N CO - 2 HN N - 2 CO2 N N N + N
H2N N O H2N N O H H - O CO - O CO2 2 N O N H H H N HN - HN N CO - N CO2 2 N N H2N N N H O OH H H2N N O H 10-formyl-tetrahydrofolate
5,10-methene-THF to 5-methyl-THF - O - O CO2 CO2 N N H H+ H HN N - HN N CO - CO2 H 2 N N N N CH2
H2N N O H2N N O H H
- 5,10-methylene-THF O CO2 reductase NADPH, FAD O CH3 N H N - HN N CO2 H H2N N N H 302 5-methyl-tetrahydrofolate
151 Purine Biosynthesis (con’t)
O NH2 aspartate, fumaric acid N GTP + GDP + Pi N NH N
PO N N PO N O O N
adenylosuccinate synthase, HO OH adenylosuccinate lyase HO OH
Inosine monophosphate Adenosine monophosphate (IMP) (AMP)
NAD+ + H O IMP 2 dehydrogenase NADH + H+ glutamine glutamate O + ATP + ADP+Pi O
N N NH NH
PO N PO N O N O O N NH2 H GMP synthase
HO OH HO OH Xanthosine monophosphate Guanosine monophosphate (XMP) (GMP)
303
IMP Dehydrogenase
O O N CONH N 2- 2 2- O3PO N O3PO N O NH + H2O O NH + NADH + H+ N N HN R O HO OH HO OH NAD+ XMP IMP Cys-S Cys-S
304
152 Pyrimidine Biosynthesis
CO2H Zn2+, O O - - H2O HCO3 + ATP Aspartic acid - - + glutamine O PO NH H2N N CO2 carbamoyl 3 2 aspartate H dihydroorotase phosphate carbamoyl synthase carbamoyl transferase phosphate
O O O HN O + HN NAD / FAD HN PRPP PO O N - - Dihydroorotate orotate CO2 O N CO2 O N CO - H dehydrogenase 2 phosphoribosyl H transferase dihydroorotate orotate HO OH Orotate monophosphate (OMP)
305
O O O HN HN HN 1) 2 ATP O O O PPPO N PO N - CO 2 PO N O O O - CO2 OMP decarboxylase HO OH HO OH HO OH
Orotate monophosphate Uridine monophosphate Uridine triphosphate (OMP) (UMP) (UTP)
ATP + GTP glutamine
Ribonucleotide CTP reductase synthase
NH2
O O N HN O HN methylene- O PPPO N O CH3 tetrahydrofolate O PO N PO N O O
thymidylate HO OH synthase HO HO Cytidine triphosphate (CTP) Thymidine monophosphate dUMP (TMP) 306
153 Ribonucleotide Reductase
PO B Ribonucleotide PO O B O Reductase
HO OH HO
Class I O H2O H2O O O O Glu Class IV Asp C Fe Fe O Class II O O O O Glu Coenzyme B12 Mn Mn µ-oxo-bridged diferric clusier
Cys S Tyr O Tyr O
Cys S Class III protein CO2 HN H3N O S Fe protein Fe S ET protein HN Fe S S A O A O O S Fe H3C
protein HO OH HO OH glycine radical 307
Mechanism of Ribonucleoside Reductase
Radical Radical-H
PnO B B P O B PnO B PnO n O O O O -H O H H H H 2
O O H HO OH O OH OH H ribonucleoside-5P H B: S Cys S Cys
B PnO B PnO PnO B O O O ET H H+ O H O H H O H S Cys S Cys S Cys S Cys S Cys
Radical-H Radical
B PnO PnO B O O H H H
HO H HO H
2'-deoxyribonucleoside-5P 308
154 Mechanism and Inhibition of Thymidylate Synthase
H H2N N N O O HN + N HN H H2N N N HN O CH3 O CH2 NHR O X 2- N O3PO O + HN NHR 2- N N O3PO O O -S Cys HO dTMP Dihydrofolate HO Glu-CO - -S Cys dUMP (X= H) 2 Glu-CO2-
309
Ternary complex of thymidylate synthase with 5’-fluoro-dUMP and 5,10-dihydrotetrahydrofolate
9,10-CH2-THF
FdUMP Tyr-108
Cys-161
pdb code: 1B02 310
155 DNA Methylation: 5-methyl-2’-deoxycytidine
NH CH3 N NH2 N 2 NH2 NH2 O C S O2C S N 2 N N N O N O N O O CH3 NH N NH3 N 3 N O O N + O O + HO OH HO OH S Cys S Cys O O
311
5-Methyl-C (Epigenetic) sequencing
NH NH2 2 O O NaHSO basic pH 3 N N NH NH sodium H2O N O O3S N O bisulfite O3S N O N O DNA DNA DNA DNA
NH2 NH2 H C NaHSO H3C 3 N 3 N sodium N O O3S N O bisulfite DNA DNA
Sodium bisulfite converst C’s to U’s, but has no effect on 5-methyl-C
312
156 Antiviral nucleosides: 2,3-dideoxynucleosides as reverse transcriptase inhibitors
Protein Biosynthesis transcription translation DNA mRNA Protein
Retrovirus (hepatitis B, HIV, HPV) ssRNA + proteins (reverse transcriptase, integrase, protease)
reverse insertion into RNA transcription ds DNA host cell genome poly-protein reverse intgrase transcriptase protease proteins
H. Temin & D. Baltimore Blackburn et al. Ch. 3.7 1975 Nobel Prize in Medicine or Physiology 313
Mechanism of action of 2,3-dideoxynucleosides: recall Sanger sequencing- termination of a growing DNA chain by enzymatic incorporation of a 2,3-dideoxynucleosides
Incorporation of a ddNTP
RT RT Termination of elongation
Truncated DNA also inhibits RT
314
157 Nucleoside-based reverse transcriptase inhibitors
O NH NH2 2 H2N O N NH N N N NH HO N N HO N HO N N OH O HO O O O O O O N O O S
N3 AZT ddC ddI d4C (-)-3TC
2,3-dideoxynucleoside is enzymatically phosphorylated at the 5-hydroxyl to the ddNTP, which is the active RT inhibitor
2- 2- O3PO3PO B B O3PO B O HO O O
ddNMP ddNDP
2- O3PO3PO3PO B O Pro-drug: administered in an inactive
ddNTP formed but is transformed in vivo into the active drug
315
DNA as a target for therapeutic agents:
Intercalation: • planar aromatic molecules “slide” between stacked bases • intercalators span the minor and major grooves of DNA • driven by hydrophobic interactions • intercalators stabilize DNA structure • causes an “unwinding” and elongation of DNA in the area of the intercalation
NH O OH O 2 O N R H3C N - Br OH N + N O H2N OCH3 O OH O N CH3 H OH O O H3C R=H, daunomycin Ellipticine NH2 Campthothecin Ethidium Bromide OH R=OH, adriamycin
316
158 Topological relationship of DNA: supercoiling, tangling, knotting for replication (transcription), the supercoiling of DNA must first be “relaxed”
Topoisomerase (mammalian) DNA gyrase (bacteria)
O Mechanism of the O B O B DNA cleavage step O HO O H+ ATP _ O P O O O B O Tyr O P O _ O _ O O B O
colvalent O Tyr enzyme-substrate intermediate Topo II inhibitors stabilize the covalent DNA•Topo II complex (protein bound double strand cleaved DNA), which causes chromosomal abnormalities and leads to apoptosis
Corbett, A.H.; Osheroff, N. Chem. Res. Toxicol. 1993, 6, 585-597 317 Topo II movies: http://berger.berkeley.edu/Pages/Teaching.html
Intercalation
major groove
major groove
minor groove
O OH O
OH
OCH3 O OH O daunomycin
O H3C
NH2 pdb code: 110D OH 318
159 Ribosomal Protein Biosynthesis:
transcription translation DNA RNA Protein (genome) (transcriptome) (proteome)
Transcription: only one of the DNA strands is copied (coding or antisense strand). Its sequence is converted to the complementary sequence in mRNA (template or sense strand), which codes for the amino acid sequence of a protein (or peptide)
RNA polymerase “splicing” DNA pre-mRNA mRNA rNTPs
319
Transcription: Intron Intron 5'-UTR Exon Exon Exon 3'-UTR Pre-mRNA:
splicing
mRNA: 5'-UTR Exon Exon Exon 3'-UTR Translation: mRNAs are transported from the nucleus to the cytoplasm, where they acts as the template for protein biosynthesis (ribosomes). A three base segment of mRNA (codon) codes for an amino acid.
320
160 Transfer RNA (tRNA): The “anticodon” region of tRNA is complementary to the mRNA codon sequence. The t-RNA carries an amino acid on the 3’-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome catalyzes amide bond formation. Although single-stranded, there are complementary sequences within tRNA that give it a defined conformation
variable loop
TψC loop
D loop
aminoacyl t-RNA 321
There are many non-standard bases found in tRNAs
O O O CH3 O O NH2 HN N N N NH NH NH NH NH CH HO HO HO HO HO N N 3 O O N O N O N O HO O N O O O N N N NH2 HO OH HO OH HO HO OH HO OH HO OH pseudouridine (ψ) dihydrouridine (D) thymidine (T) inosine (I) 7-methylguanosine 1-methyladenosine
. . . and alternative base-pairing H O H N O H N N O H N N N O H N N N N H N N N R N H N N N H O R N N R R N N R O R N I C I-A Wobble pair I-U wobble pair O O N H O O H N N H N N H O R N R N N N N N N H N N H O R N R N H N R N N O H N N H H N H R H G-C reverse pairing G-U Wobble pair A-U Hoogsteen pair
322
161 . . . and triple helix formation R N N R N N O N N O H N H H N H O H H Hoogsteen pairing H O N Hoogsteen pairing N N H N N N N N R N H N R N O N R N W-C pairing R O W-C pairing U • A-U A • A-U R N N N H N N H O H H Hoogsteen pairing N O H N
N N H N R N N N H O R H W-C pairing 323 G • G-C
tRNA Synthetase: catalyzes the biosynthesis of specific 3’-aminoacyl tRNAs from tRNAs, amino acids, and ATP
H2N O
R O
O O O N NH2 H2N O O P O P O P O N NH O N N O 2 O O O N R O P O O N N O HO OH N HO OH
O N NH2 tRNA O P O O N N O N O OH H B:
O N NH2 tRNA O P O O N N O N O OH H N ’ 2 O Class I: 2 -aminoacyl tRNAs R Class II: 3’-aminoacyl tRNAs ’ 3 -aminoacyl tRNAs 324
162 Ribosomal protein synthesis
SCH3 SCH3 OH H N H OHC N H N A site: OHC 2 O O Aminoacyl tRNA O O O O
P site:
U A C Peptidyl t-RNA U A C A U A A U G U A U G C U C U U A U G U A U G C U C U U 5' 3' 5' 3'
SCH3
OOJHC HN CH S 3 O CH3S HN OH OH OHC O OH OHC O OH N O N H H N CH OH H HN H2N CH3 HN
O OH O O OH O O
A U A
U A C
C A A U A C G A U C U G A A U A A U G U A U G C U C U U A U G U A U G C U C U U 55''' 3'' 5' 3''
E site 325
Oligonucleotide-Base Therapies: Inhibition of Protein Biosynthesis via the Antisense-Antigene (Triplex) Strategies
Taken from: “The New Genetic Medicines,” J. S. Cohen, M. E. Hogan Scientific American 1994 (Dec.), pp 75-82.
• Potentially highly selective (magic bullet) approach to therapy
• ~15 bases sequence is unique in the human genome 326
163 transcription translation protein
DNA mRNA
X X Inhibition of Inhibition of transcription translation
Hoogsteen Watson-Crick base-pairing base-pairing
327 Triple Helix mRNA•DNA
Triple Helix Motifs: third strand binds in the major groove
Pyrimidine•Purine-Pyrimidine: third strand runs parallel to the homo-purine strand H dR N N + dR T•A-T + C •G-C N N H O O H N H O Hoogsteen pairing H H H O N O H N Hoogsteen pairing N N N N H N N H N dR N N N N dR dR N O N H O dR W-C pairing H W-C pairing Purine•Purine-Pyrimidine: third strand runs antiparallel to the homo-purine strand
dR dR N N N N N A•A-T N H G•G-C N N N H H N O H H H H Hoogsteen pairing H O H N Hoogsteen pairing N N O N N N N H N N H N dR N dR N N N O dR N H O dR W-C pairing H 328 W-C pairing
164 DNA Triple Helix!
T• A- T
Major groove
C+• G- C
Minor groove
pdb code: 1BWG 329
Antisense Inhibition: inhibits mRNA function
Exon Exon mRNA Intron
splicing
Exon Exon 5'-cap AUG UAA initiating stop sequence sequence
Antisense targeting: Interon-exon splice junction: interferes with slicing 5’-cap (UTR) region: interferes with binding to the ribosome Initiation and promoter sequences: protein synthesis is not initiated Coding sequence: interferes with elongation of the protein
330
165 When an antisense oligonucleotide binds to mRNA, ribonuclease H is up-regulated. The mRNA of the mRNA•DNA hybrid is digested.
Problems with oligonucleotide-based therapies Transport: DNA does not cross cellular membranes very easily Degradation: DNA is subject to enzymatic digestion by cellular nuclease
Synthetic Oligonucleotides: must maintain affinity and selectivity and impart nuclease resistance • backbone replacements • modified bases 331
Triple Helix Oligonucleotides
HO B HO T HO O B HO O B O O
O O O HN - - + O P O S P O Me P O H2N C O B O O B O O B O HN O T
HO HO HO HO Phosphodiester Phosphorothioate Methyl Phosphonate Deoxyribonucleic Guanidines
Antisense Nucleosides Peptide Nucleic Acids (PNAs)
H3C H3C H2N n O O NH2 X NH Base N NH N N O HO N HO O N O HO O N m O O O OH
HO HO HO X=O Peptide backbone forms a helical X=S structure. Base will hybridized with ss or ds DNA or RNA with high affinity 332
166 microRNA (miRNA): genetically encoded (transcribed) but non-translated RNAs (do not code for a protein or peptide) 5'-(7-methyl-G)-
pri-miRNA
3'-(A)n- Drosha
5' ~ 70 nt 3' pre-miRNA transport to cytoplasm then Dicer
5' 3' ds RNA (21-25 nt) 3' 5'
RISC (RNAi silencing complex)
5' 3' miRNA 333
Ribonucleases (RNase): enzymes that catalyze the hydrolysis of the phosphodiester bonds of RNA
single strand specific: RNase A double strand specific: RNase III specific for DNA/RNA hybrids: RNase H
endonucleases: RNase A, RNase III exonucleoase: RNase II
334
167 A mechanism for ribonuclease A
His B B B O His O O O H N NH O His O N NH neutralization of N NH O O O O H charge makes the H N Lys O O H Lys NH3 O P - 3 phosphate more P B H O O triester like O B B P O O O O O H O O His N O H His N OH O His N OH N OH O N H O N H H
His B B B His His O O N NH O O O O H N NH H N NH O O OH O O O P O O- O H N Lys H O P - P - 3 O -O O H -O B B B His N H HO O O HO O O His N H N His N H OH OH OH N O O O H N H
335
Interference RNA (RNAi) miRNAs (or siRNAs) are important in post-trancriptional regulation of gene expression
RISC (RNAi silencing complex)- multi-protein complex with helicase and ribonuclease activity
guide strand 21-25 nt 5' 3' 5' ATP 3'
3' 5'
Ago2 5' 3' 3' 5' 3' 5' target mRNA is cleaved by RISC target mRNA
mRNA fragements degraded by RNAses; protein expression is silenced
Andrew Fire & Craig Mello, 2006 Nobel Prize in Medicine & Physiology 336 Animation: http://www.nature.com/focus/rnai/animations/index.html
168 Cellular Response to DNA Damage:
DNA Damage
Cell Cycle DNA Apoptosis Replication Arrest Repair Errors
Mutations
Cell Death Cancer
337
DNA Alkylating Agent
O NH2
N N NH NH
N N N N NH2
Cl + HN N HN N R R O Cl O Cl aziridinium ion Nitrogen Mustard active alylating agent
Nitrogen mustards will alkylate adjacent DNA bases causing DNA-DNA cross-links, which is a severe form of DNA damage and can trigger apoptosis or cause
mutations (and cancer) 338
169 Other simple DNA alkylating agents:
Alkyl halides (or reactive equvalents): dimethylsulfate methylene chloride, dichloroethane, SAM (endogenous)
Enals: acrolein (industrial chemical, cigarette smoke), 4-hydroxynon-2-enal, malondialdehyde (endogenous)
Epoxides: Butadiene diepoxide (metabolism of butadiene), chlorooxirane (metabolism of vinyl chloride), benzo[a]- pyrene diol epoxide (metabolism of benzo[a]pyrene)
339
Free-radical mediated oxidative damage to DNA Reactive oxygen species (ROS)
O O molecular oxygen
O O super oxide
H O hydroxyl radical
O O N O peroxynitrite
Causes oxidative cleavage of DNA in an O2 dependent manner The oxygen paradox: oxygen is necessary for cellular metabolism; however, oxygen is transformed into highly reactive species that can damage biomolecules. 340
170 Oxygen metabolism
pKa ~ 4.8 O2 + e O O HO O superoxide
+ 2 H 5 7 -1 -1 2 O O H2O2 + O2 k= 10 - 10 M • sec
Superoxide dismutase (SOD): Zn-Cu or Mn-Fe + 2 H 9 -1 -1 2 O O H2O2 + O2 kcat= ~ 2 x 10 M • sec
(II) (I) Enz•Cu + O2 Enz•Cu + O2 (I) (II) Enz•Cu + O2 Enz•Cu + H2O2
Catalase (heme)
2 H2O2 O2 + 2 H2O 341
Fenton reaction O (III) (II) 2 + Fe O2 + Fe
(II) (III) Redox cycling H2O2 + Fe HO + HO + Fe Very little free Fe(III) in cells Haber-Weiss reaction
O2 + H2O2 HO + HO + O2 slow
O + O O N O O O N superoxide nitric peroxynitrate oxide
pKa ~ 7.0 O + ONO2 + H HO O N HO + NO2 peroxynitrate peroxynitrous acid
342
171 Bleomycin!
Binds Fe and activates O2 giving C4 hydrogen atom abstraction mechanism of hydrogen atom abstraction may involve a Fe-O•, reminiscent of P450
+ H2N
NH N 2 H
NH2 NH H O H2N N NH2
Fe Binding O O N DNA Binding Domain N N O S Domain H O HO N H2N O NH N HN O HO S
O HO NH O N O HO O OH OH O OH
O NH2 343
Bleomycin•Co(III)OOH - DNA complex
Conformation of the DNA bound Bleomycin•Co(III)OOH complex
pdb code: 1MXK
Solution structure of the Bleomycin•Co(III) complex pdb code: 1DEY 344
172 Abstraction of the 4’-hydrogen:
5'-DNA-O3P 5'-DNA-O3P 5'-DNA-O3P O O O O B O B O2 O B RSH HO H O O O O O _ O P O _ O P O _ O P O O O O
PO3-DNA-3' PO3-DNA-3' PO3-DNA-3'
5'-DNA-O3P 5'-DNA-O P 5'-DNA-O3P 3 O O O O O B O O O H O H B: O :B O H _ _ O P O O P O _ O O O _ PO -DNA-3' O P O PO -DNA-3' 3 3 O
PO3-DNA-3'
345
Abstraction of the 5’-hydrogen
O 5'-DNA-O3P H OH 5'-DNA-O3P 5'-DNA-O P 3 O H O O O O B O B O2 O B RSH
O O O _ O P O _ O P O _ O P O O O O
PO3-DNA-3' PO3-DNA-3' PO3-DNA-3' :B H 5'-DNA-O3P O 5'-DNA-O3P O OH O B O O B O _ O P O O O _ O P O PO -DNA-3' 3 O
PO3-DNA-3'
346
173 Abstraction of the 1’-hydrogen
5'-DNA-O P 3 5'-DNA-O3P 5'-DNA-O3P O O O O B O B B O2 O RSH OH H O O O O O _ O P O _ O P O _ O P O O O O 3'-DNA-O P 3 3'-DNA-O3P 3'-DNA-O3P
5'-DNA-O P 5'-DNA-O3P 3 5'-DNA-O3P O O O O O O B O O H O H :B O _ O H O _ O P O _ _ O P O :B O P O O O O 3'-DNA-O3P 3'-DNA-O3P 3'-DNA-O3P
347
Formation of 8-Oxo-2’-deoxyguanosine and Formamidinopyrimidine Lesions by the reaction of deoxyguanosine with ROS’s
O O H O [O] N N NH H N NH NH -H+ O N O HO N NH2 N N N NH H N NH2 2 dR dR dR 8-oxo-dG [H] +H+
H H O N NH O
HN N NH2 dR
formamidopyrimidine (FAPY)
348
174 Alternative base-pairing of 8-oxo-dG during replication with pol T7
H H H H H N O H N N N O H N H O N N N H N - OR N N O OPO2PO2PO3 N H N N O N N OR N - OPO2PO2PO3 N H O O O O OH OR H OR mutagenic non-mutagenic OH
dC dA 8-oxo-dG 8-oxo-dG
349 pdb code: 1TK8 pdb code: 1TKD
DNA as a target for carcinogen: Bioactivation of pro-carcinogens:
P450 H2O
O benzo[a]pyrene
O P450
HO HO OH OH
O O O O O H O O P450 H O O H O OCH3 H O OCH3
aflatoxin B 1 350
175 O O O O O O H O O O HO H N H O HN OCH3 OCH3 O N O H2N N O HN N H t1/2 in H2O ~ 1 sec + N H2N N
O O O O O O O O O H H O O acetone H O OCH H O 3 OCH3
T. M. Harris et al. J. Am. Chem. Soc. 1988, 110, 7929
OH HO
NH2 O HO N N NH HO N N N OH N N N
351
Adducted Phosphoramidite Approach: Benzo[a]pyrene
O O N N NH NH O N N NH2 N N NH dR dR HO HO OH HO OH
N O
DMTrO O N NH solid-phase O oligonucleotide OH N synthesis N NH NH O OH AcO N N N NC P H OH O N(iPr)2 AcO C G G A C A G A A G OAc
352
176 Post-Synthetic Modification Strategy for N2-2'-Deoxyguanosine Adducts
Si(CH3)3 (H3C)3Si N O O solid-phase N DMTrO oligonucleotide N O N N synthesis N N N X X X= -F, -OTf O C G G A C A G A A G NC P O N(iPr)2
NH2 HO O OH N 1) NH HO OH OH N N N H OH C G G A C A G A A G 2) Deprotection
Kim, S. J.; Stone, M. P.; Harris, C. M.; Harris, T. M. J. Am. Chem. Soc. 1992, 114, 5480 353
Post-Oligomerization Strategy for N6-2'-Deoxyadenosine Adducts
N X X
DMTrO N solid-phase N N O N oligonucleotide N synthesis N N
O X= -F, -Cl C G G A C A G A A G NC P O N(iPr)2 OH HO
NH2 HO HO NH
1) N N HO OH N N
C G G A C A G A A G 2) Deprotection
354
177 DNA-Carcinogen Adducts! DNA-PAH Adducts: benzo[c]phenanthrene!
O N NH
N N NH dR HO
HO OH
O N NH
N N NH dR HO
HO OH
pdb code: 1HWV pdb code: 1HX4 355
DNA-Carcinogen Adducts! DNA-PAH Adducts: benzo[a]pyrene! benzo[a]pyrene benzo[c]phenanthrene
pdb code: 1Y9H 356
178 DNA-Carcinogen Adducts! MeO DNA-Aflatoxin Adduct! O O H O O H O O HO + N NH
N N NH2 dR
pdb code: 1MKL 357
DNA Repair:
1. Direct repair
2. Base excision repair (BER): repair of deglycosylation (lose of the base from the deoxyribose unit) sites, oxidation of the base, or modification by a “small” alkylating agent.
3. Nucleotide excision repair (NER): repair of “bulky” lesions
4. Mismatch Repair: repair of mis-paired DNA bases
5. Recombination: repair of double strand breaks of DNA
“Chemistry and Biology of DNA Repair” Scharer, O. D. Angew. Chem. Int. Ed. Engl. 2003, 42, 2946-2974
358
179 Direct Repair: O6-alkylguanine transferase (AGT): direct reversal by transferring the O6-alkyl group to an active site cysteine
of AGT via an SN2 reaction.
S Cys AGT H3C S Cys AGT
CH3 O O
+ N H N N NH
N N N NH2 N NH2 DNA DNA
DNA Photolyase (bacterial): direct reversal of pyrimidine- pyrimidine photodimers (UV light induced lesion)
O O O O
HN HN hν HN NH
O N O N O N N O
DNA Photolyase: FAD dependent
light dependent repair 359
O6-Alkylguanine transferase (AGT):
Cys H C Cys145 145 S 3 O S O H CH3 N N N N H H O N H H N N N H2N N O 2 H dR dR His146 His146 H N H N H O O N N H H Tyr Tyr114 114 - - Glu172 CO2 Glu172 CO2 X-ray analysis of the C145S mutant
Ser145 Tyr114
H2O
Glu172 His146 O6-Me-G
pdb code: 1T38 360
180 O6-Alkylguanine transferase (AGT): O Cys145 O covalent S S N Cys145 N protein-DNA H N N N H O N complex H N O O N O H dR dR H H His His H 146 H 146 N N O O N N Tyr114 H Tyr114 H
- - Glu172 CO2 Glu172 CO2
Tyr114
Cys145
Glu172 His146 361
Direct Oxidative Dealkylation of DNA Bases by Fe(II)-2-ketoglutarate Dependent Dioxygenases
H N 2 AlkB, Fe(II), O , H2N H2N + 2 N CH3 2-ketoglutarate + O N N N OH N N + C N – CO2, – succinate H H N N N N N DNA DNA DNA
His His His His OH O O O Asp 2 2-oxoglutarate Asp O O2 Asp O Asp O (II) Fe Fe(II) Fe(III) Fe(IV) CO (–H O) CO2 CO2 OH2 2 2 O O His His O His His OH2 OH O O 2 O O
His His His Asp Asp O Asp O O (III) Fe(III) -CO2 Fe(IV) Fe CO CO "rebound" CO 2 O 2 O 2 His O His His OH O O H2N H H N H + C 2 H N N H + C N N H N N N N DNA DNA His His Asp O Asp OH2 O (II) (II) Fe CO Fe + CO2 O 2 O His His OH2 OH2 H2N H2N + O N N OH N N + + C H H N N N N 362 DNA DNA Bugg, pg. 140-1
181 Base-Excision Repair:
O O O NH2 O O H3C H H H3C N N N N HO NH NH NH N NH NH O OHC N HN N N N NH2 N NH2 N NH2 N HO N O N O DNA DNA DNA DNA CH3 DNA DNA OGG1 Fpg AAG AAG Nth UDG
Mechanism of deglycosylation:
DNA DNA glycosylase: DNA O X O O O OH H O O O DNA H DNA O O Abasic (apurinic) site Enzyme AP lyase: leads to DNA strand scission ala Maxim-Gilbert Chemistry
DNA DNA O O X DNA DNA O H O H O OH H H O N Lys OH N H N Lys Lys N NaBH4 O Lys H DNA O O (chemical trap) O DNA O O DNA DNA
Enzyme 363
Base-excision repair: from DNA glycosylase from AP lyase
OH N Lys APE1
HO OP HO O OH CHO PO
Pol β APE1
OH OP Pol β Pol β O OH PO
HO OP
DNA Ligase
364
182 Nucleotide Excision Repair (NER): (shamefully pirated from the Scharer review)
a) A DNA lesion that causes helical distortion (red star) is initially recognized by XPC/hHR23B.
b) XPC/hHR23B recruits TFIIH to the lesion and the two helicase subunits of TFIIH, XPB, and XPD cause partial opening of the DNA around the lesion.
c) TFIIH attracts XPG and XPA/RPA to the lesion, further DNA opening takes places, and a bubble of about 25 base pairs is formed. XPC is probably no longer part of the complex at this point.
d) XPA and RPA verify the damage and ensure the proper positioning of the two endonucleases, XPG and ERCC1/XPF. XPG makes the incision 3’ to, ERCC1/XPF 5’ to the damage and an oligonucleotide of about 25–32 nucleotides in length is released.
e) The replication machinery fills in the gap and DNA ligase I seals the nick. 365
DNA Polymerases: Classified by Structural Homology A (pol I) E. coli pol I repair human pol γ mitochondrial DNA replication human pol θ repair B (α-like) human pol α priming human pol δ replication human pol ζ lesion bypass E. coli pol II repair T4 DNA polymerase phage replication C E. coli pol III replication D DNA pol D replication X human DNA pol β repair human pol λ repair Y (Umc/DinB/Rev1p/rad30 superfamily) E. coli pol IV (Din B) lesion bypass E. coli pol V (UmuDC) SOS induced lesion bypass human pol η lesion bypass human pol κ lesion bypass human pol ι lesion bypass human Rev1 lesion bypass Dpo4 archaeobacteria lesion by-pass 366
183 DNA Replication: replicative, high-fidelity DNA polymerases
Replicative DNA Polymerase
high-fidelity
Replicative DNA Polymerase
high-fidelity
DNA lesion DNA replication is blocked
367
Trans-lesion Synthesis: error prone (low fidelity), bypass polymerases (Y family)
Replicative DNA Polymerase
high-fidelity
DNA lesion DNA replication is blocked
By-pass DNA Polymerase Replicative DNA Polymerase low-fidelity high-fidelity
368
184 Xeroderma pigmentosum (XP): • genetic predisposition to sunlight induced skin cancer, as well as other abnormalities. • Inefficient repair of sunlight induced DNA lesions (XP-A-F) • DNA polymerase η is not expressed (XP-V)
O O O O
HN HN hν HN NH
O N O N O N N O
pol η T T T T A A
369
185