DNA Synthesis

3'

3'5'

SSB

5'

Leading Strand

5'

DNA Pol I

3'DNA Ligase

Lagging Strand

HelicaseGyrase

3'

5'

DNA Pol III

Primase

RNA primers DNA templateNew DNA Telomere

E. coli DNA Polymerase III

Processive DNA SynthesisThe bulk of DNA synthesis in E. coli is carried out by the DNA polymerase III holoenzyme.

• Extremely high processivity: once it combines with the DNA and starts polymerization, it does not come off until finished.

• Tremendous catalytic potential: up to 2000 nucleotides/sec.

• Low error rate (high fidelity) 1 error per 10,000,000 nucleotides

• Complex composition (10 types of subunits) and large size (900 kd)

E. coli Pol III: an asymmetrical dimer

Polymerase Polymerase

Stryer Fig. 27.30

clamp loader

Sliding clamp

3'-5' exonuclease

2 sliding clamp is important for processivity of Pol III

DNA Synthesis

3'

3'5'

SSB

5'

Leading Strand

5'

DNA Pol I

3'DNA Ligase

Lagging Strand

HelicaseGyrase

3'

5'

DNA Pol III

Primase

RNA primers DNA templateNew DNA Telomere

Stryer Fig. 27.33

Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III

DNA Ligase seals the nicks

OH P

O

-O O

O-

O P

O

O

O-DNA Ligase + (ATP or NAD+)

AMP + PPi

• Forms phosphodiester bonds between 3’ OH and 5’ phosphate• Requires double-stranded DNA• Activates 5’phosphate to nucleophilic attack by trans-esterification with activated AMP

DNA Ligase -mechanism

1. E + ATP E-AMP + PPi

OH +DNA-3' P

O

AMP-O O

O-

5'-DNA O P

O

O

O-

DNA-3' 5'-DNA

+ AMP-OH

3.

2. E-AMP + P-5’-DNA P

O

AMP-O O

O-

5'-DNA

(+)H2NP

O

O(-)

O

OH

O

Ade

ENZYME

OH

DNA Synthesis in bacteria: Take Home Message

1) DNA synthesis is carried out by DNA polymerases with high fidelity.

2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’ 3’ direction.

3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand).

4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase

Eukaryotic vs prokaryotic cells

Prokaryotes:

• no membrane-bound nucleus

• transcription and translation are coupled

Eukaryotes:

• DNA is located in membrane-bound nucleus

• Transcription and translation are separated in space and time

DNA replication in eukaryotes

Similarities with E.coli replication

1. Polynucleotide chains are made in the 5’ 3’ direction2. Require a primer (RNA).3. Similarities with the E Coli DNA Pol active site and tertiary structure

Differences

1. Eukaryotic replication is much slower (only 100 nt/sec).2. Many replication origins.3. DNA is associated with histones.4. DNA Polymerases are more specialized, and their interactions

are more complex.4. Chromosomal DNA is linear -> requires special processing of

the ends.

Eukaryotic DNA has many replication origins

Cell Cycle

Eukaryotic DNA polymerases

Size, kd

3’- exo

Function Notes

Pol

250 no chromosomal DNA replication

Inhibited by arabinosyl NTPs

Pol

39 DNA repair Inhibited by dideoxy NTPs

Pol 170 yes chromosomal DNA replication

Inhibited by arabinosyl NTPs

Pol

200 yes DNA replicationin mitochindria

Inhibited by dideoxy NTPs

Pol 260 yes DNA repair Inhibited by aphidocolin

Pol

lesion bypass

Pol

lesion bypass

Analogy between bacterial and eukaryotic proteins involved in DNA replication

Bacteria Eukaryotes

SSB RPAPol I polymerase Pol Pol III polymerase Pol 2 subunit of Pol III PCNA3’ exonuclease of Pol I RnaseH + FEN1 subunit of Pol III RCF

RPA = Replication protein A PCNA = proliferating cell nuclear antigenFEN1 = flap endonuclease

Lagging strand synthesis in eukaryotes

RPA

Pol/primase

5’

5’

(a)

(b)

5’

RNA primer

RCFPCNA

(c)

5’

Pol

(d)

(e)

Rnase H/FEN1

(f)

RPA=Replication protein A

10-30 nt

RCF = clamp loaderPCNA = sliding clamp

RnaseH = 5’-nucleaseFEN1 = flap endonuclease

ligase

Telomerase preserves chromosomal ends

• The ends of the linear DNA strand cannot be replicated due to the lack of a primer • This would lead to shortening of DNA strands after replication

RNA primer

5‘… 3'3‘… 5'

• Solution: the chromosomal ends are extended by DNA telomeraseThis enzyme adds hundreds of tandem repeats of a hexanucleotide(AGGGTT in humans) to the parental strand:

5‘… 3'3‘… 5'

AGGGTTAGGGTTAGGGTT…

telomere

5‘… 3'3‘… 5'

AGGGTTAGGGTTAGGGTT…TCCCAATCCCAATCCCAA…

RNA primer

Upstream Okazakifragment

Circular DNA does not have ends:

5‘… 3'3‘… 5'

Linear DNA:

RNA primer

Telomerase is a reverse transcriptase that uses it own RNA as a template for elongation of the 3’ end of DNA

Telomerase mechanism

Telomerase mechanism - continued

Telomeres form G-tetraplex structures

N

NH

N

N

O

NH2

N

NH

N

N

OH2N

N

HN

N

N

O

H2N

N

HN

N

N

ONH2

GG

G

G

Telomerase inhibitors

1. Telomerase RNA as a target for antisense drugs

2. G-tetraplexes at chromosomal ends as a drug target.

Porphyrins, anthraquinones: stabilize G-tetraplex structure, inhibit telomerase activity.

Modified oligonucleotides that hybridize with telomerase RNA, preventing it from beingused as a template for telomere synthesis.

Termination of Polymerization:The Key to Nucleoside Drugs

HOO

N3

N

NH

O

O

HO

NH

N

N

HN

NH2N

HO

NH

N

N

O

NH2N

O

AZT Ziagen Acyclovir

HOO

OH

OH

N

N

NH2

O

AraC

Antiviral Antitumor

Principle of action: 1) cellular uptake2) activation to 5’-triphosphate3) incorporation in DNA resulting in chaintermination

Nucleoside inhibitors of reverse transcriptase

DNA RNA Proteins Cellular Action

transcription translation

DNA

rep

licat

ion

Notable exception: retroviruses

RNA DNA

Reverse transcription

Proteins Cellular Action

translation

RNA

Typical flow of genetic information:

RNA

transcription

Reverse transcriptases (RT) are RNA-directed DNA PolymerasesUsed by RNA viruses (HIV-I , human immunoblastosis virus,Rous sarcoma virus)

1. Make RNA-DNA hybrid (use its own RNA as a primer)2. Make ss DNA by exoribonuclease (RNase H) activity 3. Make ds DNA incorporate in the host genome

RNA RNA:DNA hybrid

RT RT

ss DNA

RNAse H

RT

ds DNA

HIV Life Cycle

1 = Entry in CD4+ lymphocytes

2 = Reverse transcription

3 = Integration

4 = Transcription

5 = Translation

6 = Viral Assembly

Termination of Polymerization:Nucleoside Drugs

HOO

N3

N

NH

O

O

HO

N

N

N

HN

NH2N

HO

NH

N

N

O

NH2N

O

AZT Ziagen Acyclovir

HOO

OH

OH

N

N

NH2

O

AraC

Antiviral Antitumor

Principle of action: 1) cellular uptake2) activation to 5’-triphosphate3) competition with normal substrate and incorporation in DNA resulting in chaintermination

(zidovudine)

Other examples: dideoxycytidine, dideoxyinosine

(abacavir)

Anti-HIV drug Ziagen was discovered at the U of M College of Pharmacy

HO

N

N

N

HN

NH2N

Ziagen (abacavir)

Robert Vince, ProfessorDepartment of Medicinal Chemistry

1998

Nucleoside Drugs Must Be Converted to

Triphosphates to be Part of DNA and RNA

HOO

OH

OO

OH

PHO

HO

O

OO

OH

P

O

P

OHO

HOO

OH

Base Base

BaseO

O

OH

P

O

P

O

O

OHBase

OH

OP

OHO

HO

Kinase

Kinase

Kinase

Monophosphate

DiphosphateTriphosphate

ATP

ATP

ATP

• Compete with normal substrate for RT binding• Cause chain termination

DNA Chain termination by Nucleoside Analogs

O

OH

OPO

O

O-

3'

Template Strand

Primer Strand

Base

OPOPOP-O

OOO

O- O- O-

Mg2+

5'

Base

ZiagenNo 3’OH!

Mechanisms of selectivity

1. Activated drug is recognized and incorporated in DNA only by reverse transcriptase, not by cellular DNA polymerases (RNA viruses).

• viral polymerases usually have lower fidelity(no proofreading)

• Mammalian DNA polymerases are more accurate

2. The drug is phosphorylated by viral kinase, notby cellular kinases (e.g. AZT).

Mechanisms of resistance and possible solutions:

1. The drug cannot enter cells or is pumped out rapidly.2. The drug is rapidly deaminated to inactive form or normal substrate is

overproduced.3. The drug is no longer recognized by kinases and is not activated to triphosphate form.

Possible solution:Use activated phosphate form of nucleosides (Viread)

4. Activated drug is not incorporated in DNA by mutant reversetranscriptase (usually HIV RT mutations at codons 184,65,69, 74, and 115).

Possible solution: Use a mixture of several RT inhibitors (e.g. zidovudine (AZT) +

lamivudine (3TC) = Combivir®) or a mixture of different mechanisms of action (e.g. non-nucleoside RT inhibitors, protease inhibitors).

Nucleoside inhibitors of DNA polymerase as anticancer drugs

HOO

OH

OH

N

N

NH2

O

AraC (1--D-arabinofuranosylcytosine)

• used for treating acute myelocytic leukemia • activated to triphosphate form by cellular kinases• causes inhibition of DNA synthesis, repair, and DNA fragmentation• very toxic

DNA Damage, Mutations, and Repair

See Stryer p. 768-773

DNA Mutations

1. Substitution mutations: one base pair for another, e.g. T for G• the most common form of mutation

• transitions; purine to purine and pyrimidine to pyrimidine

• transversions; purine to pyrimidine or pyrimidine to purine

2. Frameshift mutations

• Deletion of one or more base pairs

• Insertion of one or more base pairs

Rare imino tautomer of A

N N

NH2

O

HN

NN

N

NH

C

• Very low rate of misincorporation (1 per 108 - 1 per 1010)• Errors can occur due to the presence of minor tautomers

of nucleobases.

Spontaneous mutations due to DNA polymerase errors

N

N

N

N

H2N

NH

N

O

O

AT

H3C

Normal base pairing Mispairing

10-4

amino

A(imino)T

AT

A(imino)C

AT

GC

Final result: A G transition (same as T C in the other strand)

Consider misincorporation due to a rare tautomer of A

AT

1st

replication

5’3’

Normal replication

2nd replication

Induced mutations result from DNA damage

Sources of DNA damage: endogenous

1. Deamination2. Depurination: 2,000 - 10,000 lesions/cell/day3. Oxidative stress: 10,000 lesions/cell/day

Sources of DNA damage: environmental

1. Alkylating agents2. X-ray 3. Dietary carcinogens4. UV light 5. Smoking

N

NH

NN

O

NH2

N

N

NH2

O

G C

o

h

h

HN

NH

O

ON

N

NN

OR

NH2

TO6-AlkG

n

h

G A

GC

GT

AT

Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase

DNA oxidation

H3CNH

N

O

O

H3CNH

N

O

O

HO

HO

thymine glycol

NNH

NN

O

NH2

HN

NH

NN

O

NH2

O

8-oxo-G

Reactive oxygen species: HO•, H2O2, 1O2, LOO•

•10,000 oxidative lesions/cell/day in humans

N N

NN

NH2

NNH

NN

O

NH2

N

N

NH2

O

N NH

NN

O

NNH

NH

N

O

O

NH

N

O

O

Hypoxanthine

Xanthine

Uracil

NNH

NN

O

N

N

NH

O

H

A G

Deamination

N N

NN

NH2

HO

N NH

NN

NH2HO

N NH

NN

O

Mechanism:

H2O

- NH3

A

G

C

C

Rates increased by the presence of NO (nitric oxide)

Depurination to abasic sites

N NH

NN

O

NH2O

O

O

OHOO

O

Abasic site (AP site)

H2O

N NH

NNH

O

NH2

2,000 – 10,000/cell/day

UV light-induced DNA Damage

NH

O

O

H3C

N

O

O

PO

O

O-

O

N

NH

O

O

CH3

NH

O

O

H3C

N

O

O

PO

O

O-

O

N

NH

O

O

CH3

…CC… Pyrimidine dimer

Easily bypassed by Pol (eta) in an error-free manner

Deletions and insertions can be caused by intercalating agents

Stryer Fig. 27.44

Metabolic activation of carcinogens

Stryer Fig. 27.45

N7-guanine adducts

G T transversions

carcinogen or drug (X)

X

X

mutations

replication

**intact DNA

Chemical modifications of DNA in mutagenesis and anticancer therapy

metabolic activation

repair

DNA adducts

detoxification

reactive metabolite (X-)

DNA

excretion

cell death

Anticancer Cancer

Importance of DNA Repair

• DNA is the only biological macromolecule

that is repaired. All others are replaced.

• More than 100 genes are required for DNA repair, even in organisms with very small genomes.

• Cancer is a consequence of inadequate DNA repair.

DNA Repair Types

• Direct repair– Alkylguanine transferase– Photolyase

• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair

• Recombination repair

Direct repair

• DNA photolyase (E. Coli)

NH

O

O

H3C

N

O

O

PO

O

O-

O

N

NH

O

O

CH3

NH

O

O

H3C

N

O

O

PO

O

O-

O

N

NH

O

O

CH3

5'

3'

5'

3'

N N

NN

O

NH2

CH3

O6-methylguanine

AGT-CH2-SH

N NH

NN

O

NH2

AGT-CH2-S CH3

Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)

In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.

O6-alkylguanine DNA alkyltransferase (AGT)

AGT protein is highly conserved

helix-turn-helix motif

hydrophobic side-chains form alkyl-binding pocket

Excision Repair

Takes advantage of the double-stranded (double information) nature of the DNA molecule.

Four major steps:

1. Recognize damage.

2. Remove damage by excising part of one DNA strand.

3. The resulting gap is filled using the intact strand as the template.

4. Ligate the nick.

Antiparallel DNA Strands contain the same genetic information

A ::

G :::

T ::

T

C

A

3'

3' 5'

5'

A ::

G

T ::

T

A

3'

3' 5'

5'

A ::

G :::

T ::

T

C

A

3'

3' 5'

5'

Original DNA duplex DNA duplex with one of the nucleotidesremoved

Repaired DNA duplex

Excision Repair

Takes advantage of the double-stranded (double information) nature of the DNA molecule.

Four major steps:

1. Recognize damage.

2. Remove damage by excising part of one DNA strand.

3. The resulting gap is filled using the intact strand as the template.

4. Ligate the nick.

Base excision repair (BER)

• Used for repair of small damaged bases in DNA (AP sites, methylated bases, oxidized bases…)

• Human BER gene hogg1 is frequently deleted in lung cancer

HN

NH

NN

O

NH2

O

8-oxo-G

OHOO

O

Abasic site (AP site)

NNH

NH

N

O

O

XanthineN N

NN

NH2

Me

N3-Me-Ade

Base Excision Repair

O O

O

PO

O

O-

O

O

PO

O

O-

O

O

PO

O-

O-

Base1

OH

Base3

O O

O

PO

O

O-

O

O

PO

O

O-

O

O

PO

O-

O-

Base1

Base2

Base3

O O

OH

OH

PO

O

O-

O

O

PO

O-

O-

Base1

Base3

O O

O

PO

O

O-

O

O

PO

O

O-

O

O

PO

O-

O-

Base1

Base2

Base3

R

(a) (b) (c), (d)

a) modified base is excised by N-glycosylase b) the abasic site is cleaved by AP endonuclease/lyasec) the resulting gap is filled by Polymerase b d) DNA Ligase seals the nick

AP site

Base2-ppp

BER enzyme AlkA complex with DNA

Stryer Fig. 27.48

N

N

NH2

O

NH

N

O

O

Uracil

Uracil DNA glycosylase removes deaminated C

BERC

Not normally present in DNA

N

N

NH2

O

H3CNH

N

O

O

Thymine (T)Cytosine (C)

H3C

However, deamination of 5-Me-C produces thymine:

BER

Net result: G:T base pair

Normal DNA base

No Me group

Cytosine

Nucleotide Excision Repair

• Corrects any damage that both distorts the DNA molecule and

alters the chemistry of the DNA molecule (pyrimidine dimers,

benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).

NH

O

O

H3C

N

O

O

PO

O

O-

O

N

NH

O

O

CH3

5'

3'

NH

NH

NN

NO

HO

HOOH

HOO

OH

• Xeroderma pigmentosum is a genetic disorder resulting in defective NER

Nucleotide excision repair (NER)

exinuclease

Pol /

Mammalian Enzyme

DNA ligase

Mismatch Repair Enzymes

Nucleotide mismatches can be corrected after DNA synthesis!

Repair of nucleotide mismatches:

1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base)

Parental strand is methylated:

2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)

N

N

NH2

O

H3CN N

NN

HNMe

Mismatch Repair in E. coli

Stryer Fig. 27.51

Recombination repair

DNA Synthesis in bacteria: Take Home Message

1) DNA synthesis is carried out by DNA polymerases with high fidelity.

2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’ 3’ direction.

3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand).

4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase

Genetic diseases associated with defective DNA repair

Xeroderma Pigmentosum NER

Hereditary nonpolyposis MMRcolorectal cancer

Cockrayne’s syndrome NER

Falconi’s anemia DNA ligase

Bloom’s syndrome BER, ligase

Lung cancer (?) BER