* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Types of DNA Mutations - University of Minnesota
Promoter (genetics) wikipedia , lookup
DNA sequencing wikipedia , lookup
Holliday junction wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Community fingerprinting wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Molecular evolution wikipedia , lookup
DNA vaccination wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Point mutation wikipedia , lookup
Molecular cloning wikipedia , lookup
Non-coding DNA wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
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 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 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 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 Synthesis in eukaryotes: Take Home Message 1) DNA synthesis in eukaryotes is mechanistically similar to replication in bacteria. 2) Eukaryotic replication is much slower and uses multiple replication origins. 3) Chromosomal DNA is associated with histones which need to be biosynthesized at the time of replication. 4) DNA Polymerases are more specialized, and their interactions are more complex. 5) Chromosomal DNA is linear and thus requires special processing of the ends. 6) Differences between mammalian and viral DNA polymerizes can be exploited in treatment of viral infections. 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 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: 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 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 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 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 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) 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