ucl chem2601 nucleic acids lectures

CHEM2601: Chemistry of Biologically Important Molecules Section: Nucleic Acids; Dr. Stefan Howorka

Aims of Section

Text books: DNA structure and interaction with small molecules • Free: http://www.atdbio.com/nucleic-acids-book • Nucleic Acid Structure and Recognition, Neidle, Oxford University Press, 2002, 1st edition, approx. £ 30, several copies available

in DMS Watson Library • Nucleic Acids in Chemistry and Biology, Blackburn and Gait, Oxford University Press, 1996, 2nd edition, 8 copies in DMS Watson

Library, new edition by Royal Society of Chemistry, 2005 DNA repair and DNA structure • Molecular Biology of the Gene, Watson et al, Pearson Benjamin Cummings, 2004, 5th edition, chapter 6 “The structures of DNA

and RNA” p 97-128, chapter 9 “The Mutability and Repair of DNA” p 235-258, CD-ROM with useful structural tutorials • Molecular Biology of the Cell, Alberts et al , Garland Science, 4th edition, chapter “DNA repair” p 267-275

OHO

OP

O-O O-

N

N

NH2

OOO

OH

PO

-O-O N

NH

O

O

MeDeoxycytidine 3'-phosphate;abbreviated 3'-dCMP or dCp

Deoxythymidine 5'-phosphate;abbreviated 5'-dTMP or dpT.OHO

HO

HOHO

O

HOHO

HO

NNH

N

N

NH

O

NNH

N

N

NH2

O

benzo[a]pyrene

cytochrome P-450oxidase


hydrolysis

• Know and understand structure of nucleotides, DNA and RNA at molecular level and relate structure to biological function

• Know and rationalise the chemistry and interaction of small molecules (e.g. cancerogens) and DNA and the biological consequences

http://www.atdbio.com/nucleic-acids-book






Synopsis, 1

Structure and conformation of nucleotides and oligonucleotides • Structure of nucleotides • Primary, secondary, tertiary structure of nucleic acids (L1) • Types of double helical DNA, relationship between nucleotide conformation and

tertiary structure of DNA • Deviations from ideal structures (L2) • Mismatches and mutagenesis • Triple helices • RNA structure (L3) • Structure and biological properties of modified oligonucleotides • Thermal and chemical stability of DNA duplexes


Synopsis, 2

Interactions of small molecules with DNA • Non-covalent binding: electrostatic, minor groove (L4), intercalation, biological

consequences • Covalent binding (L5): alkylating agents, metabolically activated alkylating agents • Biological consequences (L6) • Free radical and photochemical damage to DNA • Biological consequences of DNA damage; DNA repair enzymes (L7)


DNA, Deoxyribonucleic Acid is a Nucleic Acid • nucleic acids

• structure of 2-deoxy-D-ribose, a pentose sugar

• structure of heterocylic bases, aromatic structure altered by nitrogen atoms

N

NN

NH

NH2

N

NHN

NH

O

NH2 N

N

NH2

OH

N

NH

O

OH

Me

Adenine (A) Guanine (G) Cytosine (C) Thymine (T)

OHO

OH

1'

3'

5'

2'

4'OH H

CHOOHOHHOHH

CH2OH

purine bases pyrimidine bases

pentosesugar

heterocyclicbasephosphate

HCHO

OHOHHOHH

CH2OH


Nucleosides & Nucleotides • nucleosides are deoxyribose sugars linked to a heterocyclic base in the following way

OHO

OH

N

NN

N

NH2

OHO

OH

OHO

OH

OHO

OH

N

NHN

N

O

NH2

N

N

NH2

O

N

NH

O

O

Me

Deoxyadenosine

Deoxyguanosine

Deoxycytidine

Deoxythymidine

1

3

6

9

1'

3'

5' 5'

1'

3'

1

3

OHO

OP

O-O O-

N

N

NH2

OOO

OH

PO

-O-O N

NH

O

O

MeDeoxycytidine 3'-phosphate;abbreviated 3'-dCMP or dCp

Deoxythymidine 5'-phosphate;abbreviated 5'-dTMP or dpT.

• nucleotides are phosphate esters of nucleosides


Primary Structure of DNA

• single strand of DNA is formed by joining nucleotide from the 3' hydroxyl group to the 5'-OH of the next one via phosphate ester

• no 5'o5' or 3'o3' linkages (exception mRNA cap) • primary structure of DNA only determined by the

sequence of the bases • convention to write sequence from 5'o3', e.g.

dpTpApT or dTAT or TAT

5'OHP-O O

O

O

P-O O

O

NN

O

O

H

O

O

P-O O

O

O

OH

O

N

N

N

N

NHH

NN

O

O

H

T

A

T

3'


Hydrogen Bonding

• Hydrogen bonding between lone pairs of ring nitrogen and carbonyl oxygens, and –NH2 groups

• MISSING hydrogen bond donor (d) , hydrogen bond acceptor (a)

• set of complementary hydrogen bond • Watson-Crick base pairs

NN

O

NH

H

NN

N

NO

H

NHH

NN

O

NH

H

NN

N

NO

H

NHH

C G C G

:

:

:

:

:

N

N

N

N

NHH

N N

O

HO

:

:

:

A T


Factors Influencing Secondary Structure • Heterocyclic bases are key to secondary structure of DNA • Tautomerism, keto enol tautomerism, amide-imidic acid tautomerism

H3CC

H

O

H2CC

H

OH

HC

NH2

O

HC

NH

OH N

NH

O

O

H3C

N

N

OH

O

H3C

N

N

O

OH

H3C

hypothetical alternatives

N

NN

N

NH2

N

NN

N

NH2

• Lone pairs & delocalization, ring nitrogen can be protonated at N-1 o heterocyclic base

• MISSING orbitals plus lone pair

• Lone pairs & delocalization, exocyclic –l.p of NH2 lines up with S-bond, o does not protonate

• MISSING orbitals plus lone pair


Secondary Structure of DNA

O

O

P-OO

O

O

O

P-OO

O

NN

O

NH

H

NN

N

NO

H

NHH

O

O

OP-O OONH

H

NN

O

O

H NN

N

N

O

OP-O O

O

O

P-OO

O

O

OP-O

O

O

N

N

N

N

NHH

N N

O

HO O

O

OP-O OONH

H

NN

O

O

H NN

N

N

O

OP-O OO

T

C

A

T

A

T

A

5' G

3'

5'3'


Tertiary Structure of DNA

• antiparallel double helix • base pairs perpendicular to

sugar-phosphate backbone • major groove & minor groove • tertiary strucure of DNA

determined by conformational preferences of nucleotides

• minor perturbations in conformations o differently shaped DNA helices

B-DNA


Preferred Conformations of Bonds in Nucleotides • Ribose sugar ring is „puckered“ i.e. out of

plane, similar to envelope conformation of cyclopentane

• Conformation of ribose-base bond

O

O

O B2'

3'O

O

OB

2'

3'

C2' endo (C2 on the same side as base)(„S“)

C3' endo („N“)

OHO

OH

N

N

NH2

O1

3

OHO

OH

N

N

NH2

O1

3

anti syn

• Conformation about C4' – C5' bond

OO

O

B5'

4'3'

O4'H4'

PO

OROO-

R

• Rest of exocyclic bonds are determined by the preferred conformation of phosphate ester


Types of Double-helical DNA B-DNA • forms in high humidity and low salt • right-handed double helix • bases perpendicular to helix axis • sugars are C2‘ –endo • glycosidic bond, anti • major and minor grooves of same depth • phosphate backbone and both grooves

hydrated • 10 bp per turn • highly flexible

A-DNA • forms in low humidity, high salt • right-handed double helix • bases tilted 20° to enhance stacking, lie 4.5 A

away from helical axis • sugars are C3‘ –endo • glycosidic bond, anti • minor groove shallow, major groove very deep • stiff helix • 11 residues per turn

Z-DNA • left-handed double helix • Favoured for alternating G-C sequences • sugars are C3‘ –endo • purines are syn, pyrimidines anti (hence zig-zag) • minor groove narrow and deep, major groove

shallow

MBOTG, Watson Structural tutorial: DNA Structure, ABZ DNA


Deviations from „Ideal“ Structures

• predominantely affects conformations of the base pairs • dependent on DNA sequence • propeller twist, improves stacking between bases in each strand but causes steric

clash between Py(3'o5')Pu and Pu(3'o 5')Py steps

• helical twist, twist of 1 bp relative to the one below, can reduce steric clash e.g. between Me of T and the neighbouring 5'-suger in Ax/xT step

• structural deviations can make A-T rich sequences prone to unpairing and unwinding o initiations sites for transcription


Mismatches and Mutagenesis

• replication of DNA duplex is not 100% perfect • positive effect: genetic variation and role in evolution • destructive effect:mutations lead can lead to cancer • changes at the moleculer level when mutations occur (once in every 104 to 105

successful base pairing

• MISSING T A to TG mismatch leading to TA to CG transversion mutation

• transition mismatch: pairing of purine with wrong pyrimidine • transversion mismatch: pairing of two purines or two pyrimidines


Base Pair Mismatches Leading to Mutations

NNH

O

O

Me

NHN

N

N

O

H2N

thymine „wobble“ guanine

NNH

O

O

Me

NHN

N

N

O

H2NN

NH

N

N

O

NH2

NN

N

N

H2N

thymine guanine ???? „wobble“ adenine guanine base pair

recognition of wobble TG pair by endonuclease vsr, Cell 99, 615, 1999


Triple Helices • invasion of single stranded DNA into duplex

thereby forming a triplex • binding of single DNA strand does not affect

formation of base pairs in duplex • recognition of incoming strand and duplex

depends on their sequence • applications: interfere with transcription from

duplex

NHH

NN

O

O

H NN

N

N

N

N

O OH

A

T

T

T x A.T triplet

NN

O

NH

H

NN

N

NO

H

NHH

N+

N

NH

H

HO

C

C+

G

C+ x G.C triplet

triplet combine conventional Watson-Crick base pairs and Hoogsteen base pair

minor groove

major groove


RNA, Ribonucleic Acid • mRNA, tRNA, rRNA, snRNA, snoRNA, siRNA

N

NN

NH

NH2

N

NHN

NH

O

NH2 N

N

NH2

OH

N

NH

O

OH

Adenine (A) Guanine (G) Cytosine (C) Uracil (U)

Heterocyclic Bases:

D-ribose is the pentose sugarOHO

HO

1'

3'

5'

OH

OH

N

NHN

NH

O

Inosine (I) in tRNA

OHO

HO

N

NN

N

NH2Ribonucleotides

1

3

6

9

1'

3'

5'

OH

OHO

HO

N

NHN

N

O

NH2

OH

OHO

HO

N

N

NH2

O5'

1'

3'

1

3

OH

OHO

HO

N

NH

O

O

OHAdenosine Guanosine Cytidine Uridine


RNA, Primary Structure & Secondary Structure

O

O

P-OO

OH

O

O

O

P-OO

O

OH

NN

O

NH

H

NN

O

O

H

C

U

5'

3'

primary structure

O

O

P-OO

OH

O

O

O

P-OO

O

OH

NN

O

NH

H

NN

N

NO

H

NHH

O

O

OP-O OO

HO

NHH

NN

O

O

H NN

N

N

O

OP-O O

HO

O

O

P-OO

OH

O

O

O

P-OO

O

OH

NN

O

NH

H

NN

N

NO

H

NHH

O

O

O

OP-O OONH

H

NN

O

O

H NN

N

N

O

OP-O O

O

C

A

U

C

U

A

5' G

3'

RNA:RNA duplex

DNA:RNA duplex

3' 5'

3'

5' G

3'

5'


RNA Tertiary Structure

• RNA can form double helical structure, A-helix

• A-helix is assumed to be default for nucleic acids but in the case of DNA the steric hindrance of methyl group in thymine A-does not form, RNA does not have this group which would prevent A-helix

• RNA usually single stranded but can forms elements of secondary structure • RNA secondary structure elements: hairpin loop, stem, bulge, internal loop

MISSING


RNA Tertiary Structure

• tRNA, one of few examples of RNA tertiary structure, involves unusual bases and base pairs


RNA:DNA Duplex

• DNA:RNA helix more stable than DNA:DNA or RNA:RNA helix

• formed during transcription

O

O

P-OO

OH

O

O

O

P-OO

O

OH

NN

O

NH

H

NN

N

NO

H

NHH

O

O

OP-O OO

HO

NHH

NN

O

O

H NN

N

N

O

OP-O O

HO

O

O

P-OO

OH

O

O

O

P-OO

O

OH

NN

O

NH

H

NN

N

NO

H

NHH

O

O

O

OP-O OONH

H

NN

O

O

H NN

N

N

O

OP-O O

O

C

A

U

C

U

A

5' G

3'

RNA:RNA duplex

DNA:RNA duplex

3' 5'

3'

5' G

3'

5'

RNA:DNA duplex


Antisense Approach

• oligos can be made to bind tightly and prevent translation = antisense approach • to prevent degradation use of oligonucleotides with altered backbones

O

O

PMe

O

O

O

O

NN

O

NH

H

O

NN

N

NO

H

NHH

O

O

P O-O

O

NHH

O

NN

O

O

H

HO

O

NN

N

N HO

O

PO

-O

O

O

HOPO

Me

O

O

NN

O

NH

H

O

NN

N

NO

H

NHH

A

G

mRNA

G

antisense oligonucleotide:RNA duplex

OBO

PO

Me

O

OO

PMe O

OBO

OSP RP

methylphosphonate

OBO

PO

-S

O

O

phosphorothioate

OBO

PS

-S

O

O

phosphorodithioate

OBO

PO

MeO

O

O

phosphate methyl ester


Thermal stability of DNA Duplexes

• Structural stability of DNA duplexes: dissociation of DNA strands e.g. by increased temperature

• Measurement of duplex dissociation/ association by following absorbance at 260 nm • upon association A260 decreases by 25 – 30% due to formation of stacked bases and

S-S interaction • melting temperature (Tm) of DNA: midpoint of transition from duplex to single

stranded form, reflects GC content


Chemical Stability of DNA vs RNA

• primary structure of DNA is relative resistant to degration at high/low pH • DNA is resistant to base but slightly sensitive to acid at purines • RNA prone to degradation via bases

depurination

OO N

NHN

N

O

NH2

H

OP

O-O O

OO

OP

O-O O

OO

O

PO

O-O N

NH

O

O

Me

HO

OH

strand cleavage

O

OP-O O

O

O

O

B

H

O

O

P-O O-

O

O

O

B

OH

HO-H

O

O

PO O-

O

O

B

HO


Summary, 1 Structure and Conformation of Nucleotides and

Oligonucleotides

• Structure of nucleotides • Primary, secondary, tertiary structure of nucleic acids • Types of double helical DNA, relationship between nucleotide conformation and

tertiary structure of DNA • Deviations from ideal structures • Mismatches and mutagenesis • Triple helices • RNA structure • Structure and biological properties of modified oligonucleotides • Thermal and chemical stability of DNA duplexes


Synopsis, 2

Interactions of small molecules with DNA • Non-covalent binding: electrostatic, minor groove, intercalation, biological

consequences • Covalent binding: alkylating agents, metabolically activated alkylating agents • Biological consequences • Free radical and photochemical damage to DNA • Biological consequences of DNA damage; DNA repair enzymes


Interactions of Small Molecules with DNA

classification according to • non-covalent binding: electrostatic, minor groove, intercalation • covalent modification • strand cleavage

processes come about via • drugs • environmental factors

can be • therapeutic • mutagenic


External Electrostatic Interactions

• repulsion between negative charges • instable when two charges less than 7Å apart • neutralisation by cations such as Na+ or Mg2+

• A-DNA, spacing between charges 5.4 Å: high salt conditions favour compact structure

• B-DNA, spacing of 6.7 Å: favoured in low salt concentrations

• salt concentrations also affect binding of stands in duplex, parameter to regulate stringency of hybridization

O

O

P-OO

O

O

O

P-OO

O

NN

O

NH

H

NN

O

O

H

O

O

P-OO

O

O

OP-O

O

O

N

N

N

N

NHH

NN

O

O

H

T

C

A

T

5'

3'


Minor Groove Binding Agents

• major and minor grooves of DNA differ in widths, hydrophobicity, H-bonding pattern and steric effects

• small molecules such as netropsin bind to minor groove • proteins bind to major groove

NMe

O

HN NH2

NH2

NH

ONMe

NH

OHN

H2NNH2

Netropsin

NHH

NN

O

O

H NN

N

N

N

HN N

HN

HO

N

NHMe

Hoechst 33258

PDB 444D, DNA duplex minor groove binder benzimidazole derivative IB


Intercalators

• general properties of intercalators: several fused flat aromatic/heteroaromatic rings, often positively charged

process of intercalation • vertical separation of bases • aromatic groups of intercalator stack in

between • stabilisation of interaction by S-stacking, van

der Waals forces, and hydrophobic effect • results in the distortion of duplex, helix

lengthening by 3.4 Å and helix unwinding 10 to 25° depending on intercalator

• exclusion principle: no intercalation between bp next to one intercalator

• no sequence specificity

PDB 1Z3, DNA duplex intercalator ellipticine EL


Intercalating Agents Biological Consequences of Non-covalent Binding

• DNA has to unwind strands before nogalamycin can thread through

• amino sugar bind at major groove

• sugar with methoxy groups bind in minor groove

PDB 182D, DNA duplex threading intercalator nogalamcyin NGM

• biological consequences of non-covalent binders • prevent binding and activity of polymerase,

topoisomerases • e.g intercalators cause distortion of duplex and

prevent sliding of polymerases along DNA • important anti-cancer drugs


Covalent Modification of DNA, Alkylating Agents

• lone pairs in N atoms in ring and O of C=O groups of bases can react with alkylating agents • any position can react in isolated nucleoside but some positions are favoured • via SN2 mechanism: lab reagents such as MeI, MMS (MeOSO2Me), DMS(dimethylsulfate) • order of reactivity: G(N7) > A(N1) > C(N3) but also A(N3) and A(N7) • via SN1 mechanism: MNU, N-methyl-N-nitrosourea (generates CH3

+) • order of reactivity: G(O6) > T(O2) > T(O4) • accessible in DNA: G(O6), G(N7), T(O2), A(N3), A(N7)


Alkylating Agents, Nitrogen Mustards and Cisplatin

• alkylating agents mentioned so far are simple non-environmental toxins, nevertheless they are highly cancerogenic

• alkylating agents can also be of therapeutic use, anticancer compounds • nitrogen mustard derived from toxic mustard gases of World War I, used for Hodgkin

cancer treatment

B215 Coursework 2004 - model answers

N

HN N

N

O

H2N

N

NHN

N

O

NH2

N

Cl Cl

RN

Cl

R

N

HN N

N

O

H2N

N

Cl

R

N

HN N

N

O

H2N

NR

N

NHN

N

O

NH2

+

+

crosslinking of strands in duplex

CH2COOHCH3R =

• inorganic cisplatin reacting in an SN2 like substitution

N

HN N

N

O

H2N

N

Cl Cl

RN

Cl

R

N

HN N

N

O

H2N

N

Cl

R

N

NHN

N

O

NH2

+

+

PDB 1A2E, DNA duplex cisplatin CPT PNAS 1996, 93, 7606-7611


Alkylating Agents, pyrrolo[1,4]-benzodiazepines and spirocyclopropanes

P[1,4]Bi anthramycine has complicated structure but simple chemistry, loss of water produces imine which reacts with G(N2) in minor groove

N

NH3COH

O

H

HOH

CONH2 N

NH3COH

O

H

CONH2

N

HN N

N

O

H2N

N

HNH3C

OH

O

H

CONH2

N

HN N

N

O

HN

N

HN

CH3

O

N

N N

N

NH2

N

HN

CH3

HO

N

N N

N

NH2

N

HN

CH3

HO

N

N N

N

O

-H2O

-NH3

-H2O

spirocyclopropanes e.g. CC-1065 bind to minor groove of AT sequences, strained 3-membered ring reacts with A(N3)


Metabolically Activated Alkylating Agents, Trp-P-1 • pollutants that cause cancer • most are activated by endogenous enzymes to produce highly reactive alkylating agents • examples include Trp-P-1 and benzo[a]pyrene

tryptophane

heat cytochrome

P450

Trp-P-1 found in cooked and smoked meals and in cigarette smoke

acetylation in vivo

- AcOH insertion into C-H bond of dG


Metabolically Activated Alkylating Agents, Benzo[a]pyrene

• benzo[a]pyrene found in smog, car exhaust • activated form intercalates into DNA and NH2 of G reacts with epoxide

OHOHO

HOHO

O

HOHO

HO

NNH

N

N

NH

O

NNH

N

N

NH2

O

benzo[a]pyrene



hydrolysis

PDB 1XC9 polymerase bound benzo(a)pyrene DNA adduct


Biological Consequences of DNA Base Alkylation at G(N7) & A(N3)

• all of compounds mentioned so far either cause or cure cancer • mechanism by which these compounds work at the cellular level not entirely

understood

• alkylation at G(N7) by e.g. cisplatin, nitrogen mustards – can change base pairing – cross-linking of strands by G(N7) alkylation prevents replication o major cell-

killing event o used to kill cells that are dividing rapidly – alkylation at G(N7) results in depurination o apurinic site o mismatches and

target for cellular repair

• alkylation at A(N3) by e.g. CC-1065 – blocks minor groove o blocks progress of DNA polymerase – distortion of DNA o mismatches – similar effect for G(NH2)?

N

NN

NH

NH2

N

NHN

NH

O

NH2 N

N

NH2

OH

N

NH

O

OH

Me


N

NN

NH

NH2

N

NHN

NH

O

NH2 N

N

NH2

OH

N

NH

O

OH

Me



Biological Consequences of DNA Base Alkylation at G(O6)

• locks guanine into the enol tautomer o formation of wobble base pair with C

N

NN

N

O

N H

H

H3C

dG(O6Me)

N NH

OCH3

O

dT

N N

NH

H

O

dC

• forms much better base pair with T o get transition mutation from G to A o associated with cancer-causing events

N

NN

N

O

N H

H

H3C

dG(O6Me)


Radical Damage to DNA

revision of radicals • dissociation of water involves heterolytic bond fission o pair of bonding electrons is

unevenly distributed to produce proton and hydroxide ion • by contrast in homolytic bond fission one electron goes to hydrogen and the other to

oxygen o will create a H• radical and HO• radical • HO• is highly reactive and tries to remove H• from water or organic molecules such as

DNA • note difference between arrow heads

• Missing diagram


Source of Radicals • radicals generated by X-rays, J-rays, high-energy electrons, chemicals or synthetic

reagents

N

NH2H2N

Me

ONH

NH

ON

COO

NCOOCOO

N N

MeH2N

O

HNO

H2NOC HN

CONH2HH NH2

HNH

Me

HO

O

HN

HO Me

O

NH

N

S

N

SH

O

NHH

H Me

H

H

SMe2

ON

NHH

OOHHO

HOO

O OHOH

OCONH2HO

H2O HO + H

• methidiumpropyl-EDTA (MPE) intercalates into DNA, EDTA binds Fe 2+ to generate HO• radical

• bleomycin: thiazol units bind to minor groove, aminoacid derivative binds to Fe 2+ to generate HO• radical

• antitumor and antibiotic

Missing Fe2+ in above MPE circle EDTA which binds to Fe2+

MPE-Fe2+-O2 + H2O MPE-Fe3+ + HO• + HOO•

For direct attack of DNA by prehydrated electrons see JACS 131, 11320, 2009

H2O e pre

H3O+

H2O

H2O+

OH hyde

DNA

H2O


Hydroxyl Radical-Induced Strand Breaks

• 1'-hydrogen abstraction

O

O

OP

O-O O

PO

O-O B

H

O

O

OP

O-O O

PO

O-O B

O

O

OP

O-O O

PO

O-O B

O O

O

O

O

O

PO

-O O

PO

O-O

O-

PO

-O O

O-PO

O-O

OH

O2 capture

steps strand cleavage

abasic site (labile)

O

O

OP

O-O O

PO

O-O B

H

O

O

OP

O-O O

PO

O-O B

O

O

OP

O-O O

PO

O-O B

O O

O

O

O

O

PO

-O O

PO

O-O

O-

PO

-O O

O-PO

O-O

OH

O2 capture

steps strand cleavage

abasic site (labile)

O

O

OP

O-O O

PO

O-O

H

B O

O

OP

O-O O

PO

O-O

OO

BO B

COOHOPO

O-O

-OP

O-O O

OH

OH O2

steps

O

O

OP

O-O O

PO

O-O

HB O

O

OP

O-O O

PO

O-O

O OB

OPO

O-O

O

H

HOP

O-O O

OH

OH

O2

steps

• 4'-hydrogen abstraction • 5'-hydrogen abstraction


OO

O

N

NHN

N

O

NH2

HOOO

O

N

NHHN

N

O

NH2

O

Attack of Hydroxyl Radical on Guanine Base

• heterocyclic bases are extremely susceptible to attack by hydroxyl radicals • most important lesion arises when HO• attacks guanine

• bulky O atom in 8-oxo dG causes steric strain to 5' C in the usual anti conformation o syn conformation leads to better hydrogen bonding with A o transversion mutation

NN

H2N

ON

NH

HN

N

O

NH2

O

N

HN

NHN

OH2N

O

NN

H2N

O

8-oxo-G(anti) C 8-oxo-G(syn) A

N

HN

NHN

OH2N

ON

N

N

N

H2N

OO

O

N

NHN

N

O

NH2

OH

OO

O

N

NHN

N

O

NH2

HO

H- e- H


Attack of Hydroxyl Radical on Pyrimidine Bases

HN

N

O

O

MeO

OH

OH

HN

N

O

O

MeOH

O OH

HN

N

O

O

Me

HN

N

O

O

MeOH

OHN

HN

O

O

Me

OH

HN

N

O

O

MeOH

O

NHCONH2

OH

O2

and

N

N

NH2

O

N

N

NH2

O

OH

N

N

O

NH2

H

OH

N

N

O

OHH

OH

H

O

H2N

N

HN

O

O

H

OH

OH

O2

N

N

NH2

O

N

N

NH2

O

OH

N

N

O

NH2

H

OH

N

N

O

OHH

OH

H

O

H2N

N

HN

O

O

H

OH

OH

O2


Damage of DNA by UV Radiation

• exposure to UV radiation is mutagenic at low doses and cytotoxic at high doses o skin cancer

• effects are thought to arise from molecular consequences of UV radiation on DNA • especially pyrimidine bases are susceptible • photoinduced deamination of cytosine to uracil o transition mutation

• cross-linking of neighbouring thymines

HN

N N

NH

O O

O O

NNH

O

O

NNH

O

O

hv

N

N

NH2

O N

N

NH2

O

HO

H

*

N

N

NH2

OHO

hv

240 -280 nm

N

NH

O

OHO N

NH

O

O- H2O

+ H2O

- NH3N

NH

NH

OHO


Repair of Damaged DNA • details are not completely understood • two modes A) reverse the damage to the DNA B) excise the affected base • example for mode A: DNA photolyase reverse thymine dimers

HN

NH

N

NO

H2N

(Glu)n

NH

N

NH

NR

O

O

Me

Me

NH

N

NH

NR

O

O

Me

Me

NH

N

NH

NR

O

O

Me

Me

HN

N N

NH

O O

O O

HN

N N

NH

O O

O O

HN

N N

NH

O O

O O

HN

N

O

O N

NH

O

O

HN

N

O

O N

NH

O

O

MTHF

FADH-

[MTHF]*

*FADH-

*FADHe-

hv

e-

NH

N

NH

NR

O

O

Me

Me

NH

N

NH

NR

O

O

Me

Me

NH

N

NH

NR

O

O

Me

Me

HN

N N

NH

O O

O O

HN

N N

NH

O O

O O

HN

N N

NH

O O

O O

HN

N

O

O N

NH

O

O

HN

N

O

O N

NH

O

O

FADH-

[MTHF]*

*FADH-

*FADHe-

e-MTHF methylenetetrahydrofolate


Repair of Damaged DNA • O6-methylguanine-DNA methyl transferase, mode A

N

NN

N

O

NH2

CH3

S

CysHis

N NH

Pro

H

N

NHN

N

O

NH2

S

CysHis

N NH

Pro

H3C

• leads to transfer and irreversible alkylation of enzyme • methyl transferase dealkylates ethyl, 2-hydroxethyl, and 2-chloroethyl • cancerous cells develop resistance to certain types of alkylating anti-cancer drugs by

producing more copies of this enzyme


Excision Repair of Altered Base/Nucleotide

• two major pathways: base excision repair and nucleotide excision repair

ucl chem2601 nucleic acids lectures

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