Central Dogma of Molecular Biology

1. processing replication repair transcription translation DNA RNA Protein salvage synthesis dNTPs rNTPs De novo synthesis Metabolites 153B = the hottest science in town ! Central Dogmaof Molecular Biology 2009 Nobel (Medicine) 2006 Nobel (Chemistry) 2009 Nobel (Chemistry) 2006 Nobel (Medicine) 1962 Nobel (Medicine) Central Dogma Song: http://www.youtube.com/watch?v=_Q2Ba2cFAew

2. Nucleotides Structure and Synthesis 1-Structure 2-Nucleotide Synthesis Overview • Pathways Overview - De novo synthesis of ribonucleotides - Salvage of ribonucleotides • Regulation Principles • Biomedical Relevance • • Not treated: • in-depth pathways/enzymology • Nucleotides Degradation

3. A nucleotide: Pentose+phosphate+base

4. 1’ 5’ 2’ 4’ 3’ 2’ 3’ 4’ 5’ Sugar Ribose or Deoxyribose 1’ - Numbering of carbons is C1’, C2’…C5’ (‘ used to prevent confusion with the numbering of atoms in bases). - a or b configuration of the C1’ hydroxyl: b: the C1’ OH is on the same side as the exocyclic C5’

5. OH OH OH CH2 O OH CH2 O 2’ 3’ 3’ 2’ OH OH OH H Ribose Deoxyribose (2’deoxy) RNA DNA - The presence of the 2’OH confers special chemical and structural properties to RNA compared to DNA

6. Sugar puckering: C2’ endo or C3’ endo Distances between consecutive phosphate groups: 7 Å 5.9 Å C2’ endo C3’ endo • Ribose in polymers are constrained in the C3’ endo conformation for steric • reasons --> RNA is always found as C3’ endo - Deoxyriboses in DNA are in the C2’ or C3’ endo Conformation Important to remember for polymer size !

7. Formation of a phosphoester linkage O O + P O R + H2O -O OH R P OH -O -O -O Pyrophosphate Phosphoric Acid O O O P P OH O HO P OH HO Phosphates OH OH OH pKa’s important to remember for ionization of polymers at physiological pH O O Phosphomonoester P O R Phosphodiester OH P O R1 OH OH R2 O pKas = 1, 6 pKa = 1

8. H + N N H+ R2 R1 R2 R1 pKa = 3-5 Bases Aromaticity of bases and consequences: - Bases are planar • Large number of electronsin the p orbital system • Delocalization of electrons:transient dipole and • attraction between bases -> Base stacking Protonation of ring nitrogens: Ring nitrogens of bases are normally not protonated at physiological pH

9. X X Guanosine G/dG/rG Base (X=H) Adenosine A/dA/rA Nucleoside (X=pentose) Guanosine G/dG/rG Nucleotide (X=pentose phosphate) Adenosine monophosphate (AMP) Guanosine monophosphate GMP

10. Nucleoside (X=pentose) Nucleotide (X=pentose phosphate) Base (X=H) Cytidine monophospate CMP Cytidine C/dC/rC X Uridine monophospate UMP Uridine U/rU X Thymidine T/dT Thymidine monophospate TMP (dTMP) X

12. NTPs concentrations (mM) in vivo in rapidly growing yeast cells (PNAS 107, p4950, 2010) rATP 3000 dATP 16 rGTP 700 dGTP 12 rCTP 500 dCTP 14 rUTP 1700 dTTP 30 rNTPs: Used for Transcription, Signaling And general metabolism dNTPs: Used mostly For DNA replication

13. Nucleotide biosynthesis : overview de novo synthesis Salvage 7ATP 7ADP + 7 Pi PRPP + base PRPP GMP 13 reactions PPi precursors byproducts Nucleoside monophosphate recycling 12 Pi + 12ADP 12ATP 19 ATPs No ATP - Salvage is “cheap” but bases supply is limited (nuc.acids deg, foods) However this pathway is essential (Lesch-Nyhan syndrome) The only source of nucleotides in some parasites

14. de novo synthesis pathways Purines Simple metabolites PRPP 4 steps Building of the pyrimidine ring Simple metabolites Building of the purine ring onto the sugar Pyrimidines Orotate 11 steps PRPP Addition of the ring to the sugar IMP OMP UMP GMP AMP UMP kinase AMP kinase GMP kinase ADP GDP UDP Nucleoside diphosphatekinase ATP GTP UTP CTP

15. Salvage of Nucleotides PRPP+ base -> Nucleotide Enzymes specific for each base or several bases: hypoxanthine guanine phosphoribosyltransferase works with both hypoxanthine and guanine bases Salvage is most often used during conditions of cell quiescence when cells are not actively dividing Salvage of some Nucleotides is the only source of these in some parasitic organisms (Trypanosomes, ) --> enzymes are potential targets for antiparasitic drugs

16. Production of deoxynucleotides : Overview

17. The synthesis of thymidineby thymidilatesynthase TS dUMP dTMP dTDP dTTP •dUMP is the initial substrate of thymidilate synthetase (TS) •TS works close to equilibrium : [dUMP] close to [dTMP] •If TS were to use dUTP, [dUTP] would be close to [dTTP] and therefore high because of the large amount of cellular dTTP needed •The use of dUMP by TS instead of dUTP avoids the build up of large concentrations of dUTP in the cells; this would be highly mutagenic because of the use of dUTP by DNA polymerases

18. Regulation of Nucleotide Metabolism RATE of the pathways is controlled at the first committed steps • Regulation is linked to the cell cycle (G/S phases) and to the needs for nucleotides: • actively replicating cells rely mostly on de novo pathways • quiescent cells rely mostly on salvage pathways 2) BALANCE between the different types of nucleotides needs to be regulated to avoid an excess of one type of nucleotides over the other since this might be mutagenic (especially for dNTPs)

19. Nucleotide production pathways as drug targets • Principle: Actively dividing cells (cancer cells, cells from the immune system inautoinflammatory/autoimmune diseases) or viruses during viral infections have a very high demand for nucleotides (higher than “normal” quiescent, non-actively dividing cells). -> Drugs that target nucleotide production pathways will block or reduce replication of these cells/viruses and reduce their pathogenicity Examples of drugs used: • 5-FluorodUMP targets ThymidilateSynthase -blocks dTMPsynthesis • Other drugs target ribonucleotidereductase or pyrimidine synthesis (leflunomide)