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Nucleic Acid chemistry and technology

Nucleic Acid chemistry and technology. Implications of structural perturbations (hybridization) DNA synthesis and sequencing Non-enzymatic and enzymatic transformations Other functions. Nucleic acid structure can be disrupted.

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Nucleic Acid chemistry and technology

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  1. Nucleic Acid chemistry and technology • Implications of structural perturbations (hybridization) • DNA synthesis and sequencing • Non-enzymatic and enzymatic transformations • Other functions

  2. Nucleic acid structure can be disrupted • Similar to proteins, by heating, or change in pH, one can denature nucleic acid structures • Hydrogen bonds are broken, loss of base-stacking interactions cause strands of DNA double helix to separate • The strands can anneal once temperature or pH is returned to an appropriate temperature • dsDNA and ssDNA have distinct absorbance properties

  3. Melting DNA is dependent upon GC content

  4. Cyclical denaturation and renaturation of DNA is basis of PCR

  5. Hybridization is the key for microarray or “gene chip” technologies

  6. Cot analysis as a filter for repeat DNA sequences

  7. Understanding the significance of DNA sequences provides valuable insight into biology • Reactions terminated by dideoxy NTP’s

  8. Specific DNA sequences can be synthesized (i. e. primers)

  9. Alterations in DNA sequences • Purines and pyrimidines together with nucleotides undergo spontaneous alterations to their covalent structure • Deamination C  U • Hydrolysis of bond between base and pentose • UV light can induce pyrimidine dimers • Alkylating agents • Oxidizing agents • Cells have several repair mechanisms, however, permanent alterations are mutations

  10. Biosynthesis and degradation of nucleotides • Nucleotides are precursors of DNA and RNA, essential carriers of energy as ATP, GTP, NAD, FAD, CoA, etc., signaling mechanisms, and activated biosynthetic precursors. • Two pathways lead to nucleotide synthesis • de novo • salvage

  11. de novo nucleotide synthesis • Appears identical among all organisms • Bases (guanine, etc.) are NOT intermediates in pathway • Purine rings not synthesized and attached to ribose, assembled on the ribose • Pyrimidine synthesized as orotate, attached to ribose phosphate and converted to nucleotides

  12. Pyrimidines and purines share precursors • Phosphoribosyl pyrophosphate (PRPP) is a key intermediate for both (also involved in tryptophan and histidine synthesis also) • Amino acids are important precursors, glycine for purines, and aspartate for pyrimidines • Also, glutamine and aspartate serve as sources of amino groups in both purine and pyrimidine biosynthesis

  13. PRPP serves as the foundation for purine nucleotide biosynthesis

  14. Three atoms from glycine are added to the new amino group

  15. This chain is extended by formate addition

  16. An amine group is donated by glutamine

  17. The FGAM ring is closed to form AIR

  18. AIR is carboxylated to CAIR(unique because doesn’t use biotin)

  19. A mutase rearranges the carboxylate

  20. Aspartate donates an amino group in two steps to form AICAR

  21. Another formate group is donated, carried by THF

  22. Ring closure forms Inosinate (IMP)

  23. Summary of purine atom origins

  24. IMP is converted to purine nucleotides

  25. Regulation of purine biosynthesis • Three major feedback loops • Primary regualtion is AMP, GMP, and IMP inhibiting glutamine-PRPP amidotransferase (the first committed step) • Secondary regulation is inhibition of PRPP synthesis (where AMP and GMP act synergistically) • Also, regulated at bifurcation to AMP and GMP

  26. Pyrimidine biosynthesis from aspartate, PRPP, and carbamoyl phosphate • Base (as orotate) is made first then attached to ribose 5-phosphate • Orotate synthesis begins with aspartate reacting with carbamoyl phosphate to form a product which is cyclized to Dihydroorotate • Dihydroorotate is oxidized to orotate, which reacts with PRPP • This product can undergo subsequent reactions to form UMP, UTP, and CTP

  27. Pyrimidine biosynthesis regulation • Mostly through the allosteric behavior of aspartate transcarbamoylase, which catalyzes the first step and is inhibited by CTP (inhibition can be prevented by ATP)

  28. Nucleoside monophosphates are converted to nucleoside triphosphates • AMP  ADP (adenylate kinase) • ATP + NMP  ADP + NDP (nucleoside monophosphate kinases) • Nucleoside diphosphate kinase converts nucleoside diphosphates to triphosphates (generally ATP is phosphate donor)

  29. From these pathways, you note that ribonucleotides are being generated • To get deoxyribonucleotides (precursors of DNA), the 2’ carbon atom must be reduced • Accomplished by an interesting enzyme ribonucleotide reductase • A pair of hydrogen atoms originating from NADPH are passed to ribonucleotide reductase by either glutaredoxin or thioredoxin to generate an activated enzyme intermediate

  30. Ribonucleotide reductase catalytic mechanism includes free radicals

  31. Regulation of ribonucleotide reductase • Both activity and substrate specificity is modulated by binding of effector molecules • At one binding site: ATP activates enzyme; dATP inactivates enzyme A second binding site monitors substrate binding

  32. dTMP is generated from dUMP

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