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DNA Synthesis 5' 3' Helicase Gyrase Primase SSB Leading Strand Lagging Strand 3' 5' DNA Pol III DNA Pol I 3' 5' DNA Ligase RNA primers New DNA DNA template Telomere 3' 5' E. coli DNA Polymerase III Processive DNA Synthesis The 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 Sliding clamp clamp loader Polymerase 3'-5' exonuclease Polymerase Stryer Fig. 27.30 2 sliding clamp is important for processivity of Pol III DNA Synthesis 5' 3' Helicase Gyrase Primase SSB Leading Strand Lagging Strand 3' 5' DNA Pol III DNA Pol I 3' 5' DNA Ligase RNA primers New DNA DNA template Telomere 3' 5' Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III Stryer Fig. 27.33 DNA Ligase seals the nicks AMP + PPi O O OH -O P O- O O DNA Ligase + (ATP or NAD+) P O O- • 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 ENZYME 1. E + ATP E-AMP + PPi (+)H2N O P Ade O(-) O O 2. E-AMP + P-5’-DNA AMP-O P O O 5'-DNA OH OH OO 3. DNA-3' OH + AMP-O P O O 5'-DNA DNA-3' O O- + P O OAMP-OH 5'-DNA 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: Eukaryotes: • no membrane-bound nucleus • DNA is located in membranebound nucleus • transcription and translation are coupled • 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’ direction 2. Require a primer (RNA). 3. Similarities with the E Coli DNA Pol active site and tertiary structure Differences 1. 2. 3. 4. Eukaryotic replication is much slower (only 100 nt/sec). Many replication origins. DNA is associated with histones. 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 Pol 250 Pol 39 Pol 170 Pol Pol 3’- exo no Function Notes chromosomal DNA replication Inhibited by arabinosyl NTPs DNA repair Inhibited by dideoxy NTPs yes chromosomal DNA replication Inhibited by arabinosyl NTPs 200 yes DNA replication in mitochindria Inhibited by dideoxy NTPs 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 Pol I polymerase Pol III polymerase 2 subunit of Pol III 3’ exonuclease of Pol I subunit of Pol III RPA Pol Pol PCNA RnaseH + FEN1 RCF RPA = Replication protein A PCNA = proliferating cell nuclear antigen FEN1 = flap endonuclease Lagging strand synthesis in eukaryotes RNA primer 5’ (a) RPA=Replication protein A RPA Pol/primase 10-30 nt 5’ (b) PCNA RCF (c) RCF = clamp loader PCNA = sliding clamp 5’ Pol (d) 5’ Rnase H/FEN1 RnaseH = 5’-nuclease FEN1 = flap endonuclease (e) ligase (f) 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 5‘… 3‘… 3' 5' RNA primer • Solution: the chromosomal ends are extended by DNA telomerase This enzyme adds hundreds of tandem repeats of a hexanucleotide (AGGGTT in humans) to the parental strand: 5‘… 3‘… 3' 5' AGGGTTAGGGTTAGGGTT… 5‘… 3‘… 3' 5' AGGGTTAGGGTTAGGGTT… TCCCAATCCCAATCCCAA… telomere Circular DNA does not have ends: Upstream Okazaki fragment RNA primer Linear DNA: 5‘… 3‘… 3' 5' 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 N H2N N N N H2N HN N H O N O O N NH N H N O N NH2 N NH2 N N G G G G Telomerase inhibitors 1. Telomerase RNA as a target for antisense drugs Modified oligonucleotides that hybridize with telomerase RNA, preventing it from being used as a template for telomere synthesis. 2. G-tetraplexes at chromosomal ends as a drug target. Porphyrins, anthraquinones: stabilize G-tetraplex structure, inhibit telomerase activity. Termination of Polymerization: The Key to Nucleoside Drugs NH2 O NH N HN N O HO O N HO N NH N N O NH2 N HO N NH N NH2 O HO O OH O N3 OH AZT Ziagen Antiviral Acyclovir AraC Antitumor Principle of action: 1) cellular uptake 2) activation to 5’-triphosphate 3) incorporation in DNA resulting in chain termination Nucleoside inhibitors of reverse transcriptase replication Typical flow of genetic information: DNA RNA Proteins Cellular Action transcription translation DNA Notable exception: retroviruses RNA DNA RNA Proteins Reverse transcription translation transcription RNA Cellular Action Reverse transcriptases (RT) are RNA-directed DNA Polymerases Used 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 RT RT RT RNAse H RNA RNA:DNA hybrid ss DNA 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 NH2 O NH N HN N O HO N O HO N N N N O NH2 N HO N NH N NH2 O HO O OH O N3 OH AZT Ziagen (zidovudine) (abacavir) Acyclovir Antiviral AraC Antitumor Other examples: dideoxycytidine, dideoxyinosine Principle of action: 1) cellular uptake 2) activation to 5’-triphosphate 3) competition with normal substrate and incorporation in DNA resulting in chain termination Anti-HIV drug Ziagen was discovered at the U of M College of Pharmacy HN N N HO N N NH2 Ziagen (abacavir) 1998 Robert Vince, Professor Department of Medicinal Chemistry Nucleoside Drugs Must Be Converted to Triphosphates to be Part of DNA and RNA O P O HO HO HO O Base Ki nase ATP O OH OH Monophosphate ATP O O O HO P O P O P O HO OH OH O OH Base Base Ki nase O O HO P O P O HO OH ATP Triphosphate • Compete with normal substrate for RT binding • Cause chain termination Ki nase O OH Diphosphate Base DNA Chain termination by Nucleoside Analogs Primer Strand O O P Base O O- O Template Strand 3' OH O -O P O- O O P O- O O P O Base 5' O- Mg 2+ Ziagen No 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, not by cellular kinases (e.g. AZT). Mechanisms of resistance and possible solutions: 1. 2. The drug cannot enter cells or is pumped out rapidly. 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 reverse transcriptase (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 NH2 N N O HO O OH OH 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 Spontaneous mutations due to DNA polymerase errors • • Very low rate of misincorporation (1 per 108 - 1 per 1010) Errors can occur due to the presence of minor tautomers of nucleobases. H3C O H2N N NH N N N N O T amino A Rare imino tautomer of A 10-4 Normal base pairing Mispairing Consider misincorporation due to a rare tautomer of A 2nd replication 1st replication 5’ A 3’ T A T A(imino) C A(imino) T G C A T Normal replication Final result: A G transition (same as T C in the other strand) Induced mutations result from DNA damage Sources of DNA damage: endogenous 1. Deamination 2. Depurination: 2,000 - 10,000 lesions/cell/day 3. Oxidative stress: 10,000 lesions/cell/day Sources of DNA damage: environmental 1. Alkylating agents 2. X-ray 3. Dietary carcinogens 4. UV light 5. Smoking Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase O N N o h N NH h N NH2 O G OR n N N NH2 N N N O NH HN h NH2 O O6-AlkG C T G A G C G T A T DNA oxidation Reactive oxygen species: HO•, H2O2, 1O2, LOO• O O H3C H3C HO NH N O N NH N N O thymine glycol O H O N HO NH NH2 N NH O N N NH2 8-oxo-G •10,000 oxidative lesions/cell/day in humans Deamination NH2 N O N N N N N A O N N G N NH2 O N N Mechanism: Hypoxanthine N HO O N N NH N NH2 NH NH2 NH N H Xanthine N O N N H2O N - NH3 N O HO NH2 N NH NH O N C O N N N Uracil H NH A NH N N O O G N C Rates increased by the presence of NO (nitric oxide) N NH N Depurination to abasic sites O N O O O N O NH N H2O NH2 O O OH O Abasic site (AP site) 2,000 – 10,000/cell/day N N H NH N NH2 UV light-induced DNA Damage O H3C O NH NH N O H3C N O O CH3 O NH O O P OO N O …CC… O O O CH3 NH O O P OO N O O 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 N7-guanine adducts G T transversions Stryer Fig. 27.45 Chemical modifications of DNA in mutagenesis and anticancer therapy carcinogen or drug (X) detoxification metabolic activation excretion reactive metabolite (X-) DNA DNA adducts X X repair replication * intact DNA cell death Anticancer * mutations 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) O O H3C H3C NH NH N 5' N O 5' O CH3 O N O CH3 O NH O O P OO O NH O O P OO N O O 3' O 3' O O6-alkylguanine DNA alkyltransferase (AGT) 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. O N N CH3 O N N N NH AGT-CH2-SH NH2 O6-methylguanine N N AGT-CH2-S NH2 CH3 AGT protein is highly conserved hydrophobic side-chains form alkyl-binding pocket helix-turn-helix motif 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 5' 3' 5' 3' 5' 3' 3' A :: T A :: T A :: T G ::: C G G ::: C T :: A T :: A T :: A 5' Original DNA duplex 3' 5' DNA duplex with one of the nucleotides removed 3' 5' 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…) H N O N N O O O OH N NH NH O 8-oxo-G O NH2 Abasic site (AP site) N NH2 N H O N Xanthine N N N Me N3-Me-Ade • Human BER gene hogg1 is frequently deleted in lung cancer Base Excision Repair Base2-ppp O O O Base1 O O P OO O Base2 O O P OO O O O P OO- O O O P OO O R O Base1 Base1 O OH O O P OO O (b) Base3 O O P OO- O Base1 O O P OO O OH (a) Base3 O Base2 (c), (d) OH O P OO O Base3 O O P OO- O O P OO O O O P OO- AP site a) modified base is excised by N-glycosylase b) the abasic site is cleaved by AP endonuclease/lyase c) the resulting gap is filled by Polymerase b d) DNA Ligase seals the nick Base3 BER enzyme AlkA complex with DNA Stryer Fig. 27.48 Uracil DNA glycosylase removes deaminated C No Me group NH2 O N N BER C NH O N Cytosine O Not normally present in DNA Uracil However, deamination of 5-Me-C produces thymine: O NH2 H3C H3C N N O Cytosine (C) NH N BER O Thymine (T) Net result: G:T base pair Normal DNA base 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). O H3C O NH N 5' O O O P OO N O HO32N CH Pt NH H2N N Cl Cl O O 3' • HO O NH H2N N OHNH O N 2 HOPt OH H2N OH2 HO OH H N NH2 N -GGH2N H2N N Pt N O N N NH N NH2 Xeroderma pigmentosum is a genetic disorder resulting in defective NER Nucleotide excision repair (NER) Mammalian Enzyme exinuclease Pol / 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: H3C NH2 HN N N N O N Me N N 2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt) 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 colorectal cancer MMR Cockrayne’s syndrome NER Falconi’s anemia DNA ligase Bloom’s syndrome BER, ligase Lung cancer (?) BER