BMSC 804 Nucleic Acids-structure_synthesis_chemotherapy Fall 2024.pptx

Nucleic Acid Biochemistry Information Metabolism Nucleic Acids DNA/RNA structure Nucleotide synthesis Chemotherapy BMSC 804 – Fall 2024 David J. Samuelson, PhD [email protected] 1 Information Metabolism Central Dogma...

Nucleic Acid Biochemistry Information Metabolism Nucleic Acids DNA/RNA structure Nucleotide synthesis Chemotherapy BMSC 804 – Fall 2024 David J. Samuelson, PhD [email protected] 1 Information Metabolism Central Dogma Of Molecular Biology Replication DNA Reverse Transcription Transcription RNA Translation Protein Post-translational modification Maturation 2  Prokaryotic & Eukaryotic Cells Many intrinsic biochemical processes that developed in early Prokaryotic life forms on Earth have Cell changed little as organisms evolved to become more complex Eukaryotic Cell 3  IMPORTANT What does a indicate? Important material/information to learn for comprehending biochemistry and molecular biology Learning Objectives Molecular structure, along with chemical and physical properties of nucleic acids that make storage and accessibility of genetic information possible Biochemical principles and enzymes of nucleotide metabolism Chemotherapeutic mechanisms of antimetabolite and DNA damaging drugs A continuous thread throughout is to acquire comprehensive knowledge of how all organisms use similar or highly conserved mechanisms of information storage and metabolism 4  Nucleic Acids - Relevance to Life Nucleic acids are molecules of INFORMATION!! Reproduction, Development, Function, & Evolution Genetics Genetic Mutation/Variation – Generational inheritance – Species adaption Development – Speciation – Species integrity Function – Organism maintenance  Nucleic Acids Two Types Ribonucleic acid (RNA) Deoxyribonucleic acid (DNA) Polymer chains with similar monomer units  Monomer units of DNA and RNA: Nucleotides 5' Nucleic acids contain 1. Pentose sugar (ribose, 2’-deoxyribose) 3' 2' 2. Phosphate (mono, di or tri) 3. Base (purine or pyrimidine) Pentose Sugar ribose Nomenclature: in RNA nucleotide = sugar + base + phosphate nucleoside = sugar + base Numbering: Base: numbers Sugar: primed numbers (3') Phosphates: a, b, g 7  RNA/DNA: Two types of sugar backbones 1. Nucleotides in DNA/RNA (Sugar, phosphate, base) a. Sugars: Two types: Ribose (RNA); 2’-deoxyribose (DNA) 2. Phosphate(s): mono, di, tri NTP or dNTP 2’-deoxyribose (all four nucleotides) 8 Two types of bases in DNA & RNA: Purines & Pyrimidines 1. Purines (A,G) (DNA/RNA) (DNA/RNA) 2. Pyrimidines (C,U,T) 9 (DNA/RNA)  Phosphodiester bonds link nucleotides to form nucleic acids 5’ end Free phosphate 2’-deoxyribose Free H H ribose 10 hydroxyl 3’ end  Nucleic Acids Strong Acids Phosphate group pKa ~ 1 Nucleotide  Nucleic Acid (DNA & RNA) Structure A. Nucleic acids are polymers of nucleotides a. DNA is polymer of deoxyribonucleotides b. RNA is polymer of ribonucleotides. B. DNA is most often used as the genetic material Stability Copying mechanisms exception: RNA viruses C. DNA Structure: a. Polarity: nucleotides are joined by a 5'-3' phosphodiester linkage. By convention DNA and RNA are written in the 5’ -> 3’ direction. b. The backbone of the nucleic acid strands are alternating pentose and phosphates (sugar phosphate). 12 DNA Double Helix Structure 13  DNA Structure - Double Helix DNA is double stranded: Two polymer strands 5’ 3’ running anti-parallel, these strands are complementary. B form DNA is most common. It is a right- handed double helix, bases are stacked 0.34 nm apart, each base pair is set 36 o from the previous base so that 10 base pairs make a full turn. 3’ 5’ DNA has major and minor grooves. www.anselm.edu/.../genbio/doublehelix.JPG 14  DNA Double Helix Forms Variables Base sequence Solvent composition Can bend (AT rich) A (right) B (right) Z (left) 15  DNA Double Helix c. Chargaff’s rule: amount purines (AG) = pyrimidines (CT) and A=T, G=C d. Double Helix held together by hydrogen bonds between complementary base pairs. Force stabilizing nucleic acid structures G-C: 3 hydrogen bonds A-T : 2 hydrogen bonds 16  DNA can form secondary structures besides a double helix Other secondary structures of DNA likely have a mechanistic role in cells tetraplex or quadruplex DNA aptarmers (short oligonucleotides) 17  Physical properties of DNA DNA can “melt”. DNA “melts” or denatures (becomes single stranded) when temperature is increased DNA can Anneal or “hybridize” The double stranded form is energetically more favorable under mild conditions, so double helix reforms spontaneously. Complementary strands reanneal. Nucleic acid hybridization is a property that makes much of what is possible in biology and 18  Base composition and DNA Base composition affects melting temperature. The term used is Tm = the temperature at which half the DNA is denatured to a single stranded state. Hyperchromic effect striking absorbance Tm increases as DNA 19 denatures or melts Biological Consequences& Biotechnical Utility of DNA Structural Properties 1. Each strand is template for other strand – DNA replication & DNA repair. 2. DNA is used to store, disseminate, and pass on information. 3. Nucleic acid sequences can be determined Amount of DNA in a human somatic cell 6,320,000,000 base pairs in a diploid human cell. That is 6.32x109 bp x0.34nm/base= ~2.15 meters. Partitioned into 46 chromosomes, 22 autosomal pairs plus XX or XY sex chromosome pairs. How is all that DNA stored in a cell? Supercoiling and extreme packaging 20  How is DNA packaged inside a nucleus? this is achieved by wrapping DNA around a protein core. This base unit is a nucleosome Chromatin = DNA + protein core Protein core made up of histones core histones: H2A, H2B, H3, H4 Nucleosome: Core Particle linker histone: H1 8 histones + 146 bp DNA Histones are highly basic (positively charged) Chromatosome: 9 histones + 200 bp DNA 21  Chromatin Structure 1. Supercoiling of Chromosomal DNA starts with nucleosomes, but there are increasing levels of supercoiling to produce chromatin. DNA supercoiled by enzymes called topoisomerases: 2 types Type I: break one strand-relax DNA Type II: break both strands- supercoil-ATP Topoisomerases are targets for cancer drugs. Topotecan, Irinotecan. Inhibit type I topoisomerases. ovarian, lung and colorectal cancer 22  Chromosome features 1. Telomeres: physical ends of chromosomes (TTAGGG)n, with 3' single stranded extension. 2. Centromere: DNA sequence of a chromosome that attaches to the mitotic spindle so that daughter cells contain the correct number of chromosomes. 3. Origin of replication. Where DNA synthesis starts during replication. 4. Karyotype: chromosome makeup of a cell. 23  Nucleotide Synthesis Nucleotides are the monomers of nucleic acids Purine & Pyrimidine Biosynthesis Deoxyribonucleotide (dNTP) Biosynthesis Nucleotide Salvage & Catabolism 24 Two types of bases: Purines & Pyrimidines 1. Purines (A,G) (DNA/RNA) (DNA/RNA) 2. Pyrimidines (C,U,T) 25 (DNA/RNA) Base + Sugar = Nucleoside Base + Sugar + Phosphate = Nucleotide = …ate 26  Metabolic Sources of Nucleotides: A. De novo biosynthesis : Almost ALL organisms synthesize nucleotides Purines:amino acids, bicarbonate, tetrahydrofolate, energy Pyrimidines: amino acids, bicarbonate, energy. B. Salvage – Turnover or Recycling of cellular nucleic acids Digestion of ingested nucleic acids 27  Nucleotide Synthesis PRPP synthetase: enzyme responsible for the synthesis of activated ribose (5'- phosphoribosyl-1'-pyrophosphate, PRPP), which is necessary for de novo synthesis of purine and pyrimidine nucleotides. PRPP synthetase is aka Ribose- phosphate pyrophosphokinase and Ribose-phosphate diphosphokinase PRPP is required for synthesis of both Purines and Pyrimidines (and nucleotide salvage) 28  - Nucleotide Synthesis – a common starting point PRPP Synthetase Pyrimidine Purine Synthesis Synthesis 29 Purine Nucleotide Synthesis From R5P to Inosine monophosphate (IMP) 11 Rxs Base building Figure 23-1 Voet, Donald; Voet, Judith G.; Pratt, Charlotte W.. Fundamentals of Biochemistry: Life at the Molecular Level, 5th Edition (p. 804). Wiley. Kindle Edition. 30  Purine Biosynthesis. 1. Purines (Adenine, A; Guanine, G) a. PRPP – activated sugar HC03- b. Amino acids provide Ns. glutamine 2N aspartate 1N glycine 1N , 2C c. HC03- (bicarbonate), 1C d. Tetrahydrofolate, 2C e. Energy (ATP) 31  Purine Biosynthesis. 2 Committed step Inosinate OR Many steps Inosine mono- phosphate (IMP) base is First nucleotide ! IMP (Inosinate) hypoxanthine 32  Purine Biosynthesis. IMP converted to A, G AMP IMP GMP 33  Production of nucleotide di- and tri-phosphates A. Base-specific nucleoside monophosphate kinases synthesize nucleoside diphosphates Adenylate kinase ATP + AMP 2 ADP Guanylate kinase ATP + GMP ADP + GDP B. Nucleoside diphosphate kinases convert nucleoside diphosphates to nucleoside triphosphates. These enzymes do not discriminate between ribose and deoxyribose. ATP + NDP (dNDP) ADP + NTP(dNTP) 34 Purine Nucleotide Biosynthesis is Regulated IMP Pathway Control Points 1 Feedback Inhibition ADP and GDP Feedforward Activation 2 Allosteric activation by PRPP Branch Point Control Rates of AMP and GMP are coordinated AMP & GMP are competitive inhibitors of IMP 35 Metabolic Disorders in Purine Biosynthesis Broad clinical spectrum – Neurological impairment: psychomotor retardation, epilepsy, hypotonia, microcephaly – Sensory involvement: deafness, visual disturbances – Multiple malformations Muscle presentations Hyperuricemia: gouty arthritis, kidney stones See Review by: Dewulf et al. Molecular Genetics and Metabolism 136:190-198, 2022 36  Purine Ribonucleotide Synthesis Summary – Key Points Purines are synthesized by assembling a purine base on 5'- phosphoribosyl-1'-pyrophosphate (PRPP), a molecule produced from Ribose-5-phosphate (R5P) and ATP IMP is the first purine nucleotide synthesized. PRPP, amino acids, folate, and ATP are used to synthesis purines AMP and GMP are synthesized from IMP. The biosynthesis of ATP and GTP are reciprocally regulated by the concentration of the other Kinases convert AMP and GMP to ATP and GTP Purine synthesis is regulated by feedback inhibition and feedforward activation to maintain a concentration of purines. Branchpoint control regulation is used to maintain a balance of each purine 37 Pyrimidine Biosynthesis 6 Steps to UMP 38  Pyrimidine Biosynthesis. De novo Biosynthesis Ingredient List Glutamine Aspartate HC03- (bicarbonate) Energy (ATP) PRPP – activated sugar Carbamoyl phosphate 39  Pyrimidine Biosynthesis. CAD protein is a trifunctional 2ATP,CO2 enzyme in animal cells that Glutamine catalyzes the first three steps of pyrimidine biosynthesis Carbamoyl phosphate Synthetase II 2ADP, Pi, glutamate Carbamoyl phosphate Aspartate Committed Aspartate step transcarbamoylase Pi Carbamoylaspartate Dihydroototase L-Dihydroorotate 40 Orotate Pyrimidine Biosynthesis (U and C). First pyrimidine ODCase: OMP- decarboxylase makes UMP from OMP CTP Glutamine + ATP OMP UMP UTP Kinases 41 Pyrimidine Biosynthesis - CTP from UTP Animals use glutamine Bacteria use ammonia (NH3) to gain the amino group 42  Production of nucleotide triphosphates A. The nucleotide triphosphates, which are used in RNA synthesis, are produced by nucleotide kinases (use ATP) Nucleoside monophosphate kinases ( generally specific for a particular base) ATP + NMP ADP + NDP B. Nucleoside diphosphate kinase ( not specific to a particular base, either purine/pyrimidine or ribo/deoxyribo) add third phosphate group ATP + NDP (dNDP) ADP + NTP(dNTP) 43 Regulation of Pyrimidine Synthesis Carbamoyl phosphate Synthetase II regulated in animals Activated by ATP, PRPP Inhibited by UTP, UDP Allosteric regulation Regulatory molecule binds the regulated enzyme at a site other than the enzyme’s active site ATCase regulated in bacteria OMP decarboxylase (ODCase) competitive inhibitors are UMP and CMP 44  Disorders of Pyrimidine Metabolism Clinical features – Developmental delay – Seizures – Ataxia – Language deficits – Mental retardation – Anemia – Hypouricosuria – High levels of uracil and thymine in urine 45  Pyrimidine Synthesis Summary – Key Points Pyrimidine nucleotides are synthesized by placing an assembled pyrimidine base on 5'-phosphoribosyl-1'-pyrophosphate (PRPP) OMP is the first pyrimidine nucleotide synthesized UMP is synthesized from OMP & CTP is made from UTP, glutamine, ATP, & H2O Kinases convert UMP to UTP Pyrimidine synthesis is regulated by substrate, (PRPP) and energy (ATP) levels, (activated) and product (UDP, UTP) levels (inhibited) TMP, TDP, or TTP are not synthesized (thymine nucleotides are only synthesized as 2’-deoxy forms) 46  Convergence of Purine & Pyrimidine Nucleotide Biosynthesis to make dNTPS RR RR = Ribonucleotide Reductase (RR): one enzyme produces 2’-deoxy (d) ribonucleoside diphosphates: dADP, dGDP, dCDP, and dUDP 47  Synthesis of 2’-deoxy-ribonucleotides for DNA a. Catalyzed by ribonucleotide reductase (RR). b. NDP is reduced (2’ position) to form the dNDP. c. NADPH provides reducing power, via a protein intermediate, thioredoxin. (glutaredoxin can be used in place of thioredoxin) Ribonucleotide Reductase NDP (G,A,C,U) ---------------- dNDP 48 Regulation of dNTP Synthesis Goal of ribonucleotide reductase enzyme regulation is to produce correct ratios of the 4 dNDPs 49 http://en.wikipedia.org/wiki/File:Rnr.jpg 50  Purine & Pyrimidine Biosynthesis RR 5 & 6 Together make dTTP from dUMP 51  Production of Deoxythymidine – for DNA only a. Add methyl group to dUMP b. Methyl donated by N5,N10 –methylene- tetrahydrofolate c. Catalyzed by thymidylate synthase d. No ribothymidylate so dTMP, dTDP, dTTP used, always 52  Folate derivative acts as 1 carbon donor Sources dUMP N5,N10 –methylene-tetrahydrofolate must be regenerated by Dihydrofolate reductase, a key enzyme in the biosynthetic production of dTMP. 53 Nucleotide salvage and degradation Nucleotide salvage (reuse of bases) Nucleotide degradation (catabolize bases) SOURCES: Cell Turnover Dietary Nucleic Acids Pathogens 54  Nucleotide Metabolism Dietary nucleic acids Stomach Intestine – pancreatic nucleases & intestinal phosphodiesterases yield nucleotides Nucleotides are ionic – cannot pass through cell membranes Nucleotidases and phosphatases yield nucleosides Nucleosides are absorbed through the intestine or further degraded by nucleosidases and nucleoside phosphorylases 55  Nucleotide Degradation Purines are broken down to uric acid Uric acid may be further catabolized for excretion Pyrimidines are converted to CoA derivatives for catabolism √ Nucleotide Metabolism 56  Nucleotide degradation and salvage Purine degradation (end product Uric acid) a. Degradation proceeds by removing base from nucleotides, then sugars. b. Deficiency of adenosine deaminase causes SCIDs (bubble boy, gene therapy) Purine Salvage (recycling of bases) a. Bases added to PRPP by HGPRT (HPRT) b. Deficiency of HGPRT (HPRT) causes Lesch- Nyhan syndrome, lethal disease. -Uric acid crystals -feedback inhibition PRPP synthase causing deficiency in all nucleotides 57  Purine Salvage Free purines are reconverted to corresponding nucleotides Adenine phosphoribosyltransferase (APRT) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Lesch-Nyhan syndrome: mutations resulting in severe HGPRT deficiency 58  Purine degradation and salvage Biosynthesis Salvage Salvage Salvage (base reused) (base reused) HGPRT = HPRT Hypoxanthine (Guanine) Phosphoribosyl Uric Acid transferase Degradation Urate 59  Gout Purines are broken down to uric acid Gout Excess of uric acid Allopurinol treats gout by irreversibly inhibiting xanthine oxidase Xanthine oxidase 60  Gout: Allopurinol inhibits uric acid synthesis GMP AMP Xanthine inhibitor Used to prevent gout attacks. Oxidase Xanthine Oxidase Urate 61  Nucleotide degradation and salvage Pyrimidine degradation or salvage a. Degradation to urea, via various intermediates b. Salvage does occur, but enzymes involved have high Km orotic acid (or uracil) + PRPP -> pyrimidine nucleotide pyrimidine + ribose-1-phosphate -> pyrimidine nucleoside NOTE: physiological relevance not clear (high Km) Important when giving pharmacological doses of pyrimidine analogs because they get incorporated into DNA and kill cells. 62  Dental Biochem '16 Cancer Therapy Surgery Antimetabolite Radiation therapy Immunotherapy Chemotherapy – Cytotoxic Competitive Inhibitor Hypoxanthine-guanine Kills dividing cells phosphoribosyltransferase – Targeted (HGPRTase) Kills cancer cells by targeting molecular properties unique to cancer cells 63  Chemotherapy* *Chemotherapy is a broad and expanding field that refers to treating any disease with a drug. This topic will be limited, during this course, to DNA damaging and antimetabolite drugs that work by inhibiting nucleotide and/or nucleic acid synthesis. You may be interested in learning more about other cancer therapies: For example, targeted cancer therapies that include immune checkpoint inhibitors  Dental Biochem '16 Antimetabolite Drugs: not just for cancer therapy Drug Use 6-Mercaptopurine Acute Lymphocytic Leukemia (ALL) Azathioprine Immunosuppressant Allopurinol Gout Acyclovir Antiviral Methoprim Antibacterial Azidothymidine (AZT) AIDS Cimetidine Stomach ulcers Propranolol High blood pressure Fluorouracil Anticancer Methotrexate Anticancer Autoimmune 65 Chemotherapeutic Drugs – antimetabolite class  5 Fluorouracil 5-Fluorouracil - Antimetabolite used to treat cancer. (5FU) 5F-Uridine-inhibits thymidylate synthase Drug administered must be converted to 5F- dUMP to be efficacious Used to treat carcinomas of GI tract, breast, and ovarian cancer. ? What is the effect of the Fluorine atom? Is there a biochemical difference between 5Methyl-dUMP and dTMP? 67  5 FU and methotrexate inhibit synthesis of dTMP Both 5-fluorouricil (5-FU) and methotrexate act Binds covalently by blocking to enzyme synthesis of dTMP 68  5-FU Metabolism FUMP RNA OPRT RR 5F-dUMP DPYD RR OPRT = orotidylate phosphoribosyl transferase ? Multi-drug cocktails are used to treat cancer.  DNA damage response Apoptosis is programmed cell death http://web.mit.edu/beh.109/www/Module3/lectureslides/BE109%20Module%203%20Day%203%20Lecture.pdf 71  Chemotherapeutic Drugs DNA damaging agents Nucleophilic centers are electron-rich regions that are available to participate in addition and substitution reactions The N7 of Guanine is the Nucleophilic most nucleophilic site of Centers in DNA DNA bases Friedberg et al, Fig. 2-30 Alkylating Chemotherapeutics Friedberg et al, Fig. 2-29 73  Interstrand Crosslinks Nitrogen Mustard Friedberg et al, Fig. 2-31 Crosslinks between complementary strands of DNA 2017 74  Why are approved DNA damaging agents better at killing cancer cells than non- cancer cells? Therapeutic Index Chemoresistance Mechanisms Decreased drug uptake Increased drug export Increased drug detoxification Reduced activation of pro-drug Increased DNA repair 76  DNA repair pathways Common Damaging Agent Direct Repair Alkylating agents Base excision repair Alkylating agents Nucleotide excision repair UV light Mis Match Repair DNA replication error Single strand break Repair Ionizing Radiation Double strand break Repair http://www.hpa.org.uk/radiation/publications/misc_publications/dna_database/dna_database_main_text.pdf 77  Ionizing Radiation (IR ) DNA Damage: Strand Breaks & ROS High LET – α particles, cosmic rays – dsDNA breaks (PLD†) Single- Low LET* strand – breaks ß particles, γ-rays, x-rays caused by – ssDNA breaks x-rays, for – Base damage example – ROS http://jolisfukyu.tokai-sc.jaea.go.jp/fukyu/mirai-en/2007/6_5.html *LET = Linear Energy Transfer † PLD = Potentially Lethal Damage 78  Double-Strand DNA damage Both strands have damage! Damaged Chromosome Non-damaged Homologous Chromosome Chromosomes exist as pairs in somatic cells A non-damaged chromosome can be used to repair its double-strand DNA damaged homologous chromosome 79 Homology Directed Repair (HDR ) of Double- Strand DNA Damage BRCA1 & 2 Uses recombination between DNA of the undamaged and damaged homologous chromosomes to repair the damaged chromosome. Inherited mutations in BRCA1 or BRCA2 greatly increase lifetime risk of developing breast and ovarian cancer 80  What happens when high-fidelity, homology-directed repair is not available? Non-Homologous End Joining (NHEJ) repair Mammalian NHEJ Smart & Hodgson, Molecular and Biochemical Toxicology, 2008 BIOC675 Cancer Biology 81  Study Guide Learn biochemical structures and features of nucleotides and nucleic acids. Understand physical properties of DNA and why these are relevant to molecular biology. Learn how nucleotides synthesized, degraded, and salvaged. Learn general mechanisms of action of antimetabolite and DNA damaging drugs. Learn and understand the biochemistry of mechanistic pathways of specific drug examples covered in class. Learn that DNA damage is repairable and failure to repair DNA damage is one chemotherapeutic mechanism used to destroy cancer cells 82