GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL A two (2) unit course for non-biochemistry majors only.

Transcription

1 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 A two (2) unit course for non-biochemistry majors only. PREREQUISITES: Biology 181 Organic Chemistry (Chem 241a,b) Concurrent/previous registration in Biochemistry 460 COURSE CONTENT: Mechanisms of DNA replication Gene transcription and translation Recombinant DNA Regulation of gene expression Molecular biology of viruses, including avian flu and HIV LECTURES: Tuesday and Thursday, 11:00-11:50 AM, Bio. Sci. East, Room 100 Lecture materials available on web as notes, illustrations, etc. Some Lecture material will be learned from online notes, study questions, in-class and other discussion i interactions ti with instructors, the textbook and some assigned reading.

2 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 INSTRUCTOR: Dr. Don P. Bourque Office Hours: [After lecture, Tuesday 2-4:00 PM by appointment] Exam Reviews [Dates TBA on Lecture Schedule, Room TBA] TEACHING ASSISTANT: Michael Pham Office Hours: [BSW 251A] Tues, 8:30-10:30 AM Reviews, Discussions, Extra credit Presentations: [Tuesday 5:30-6:30 PM, Room TBA]

3 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 TEXTBOOK: Biochemistry, J.M. Berg, J.L. Tymoczko, L. Stryer, 6th Edition, W.H. Freeman (2007) CLASS MATERIALS: All available at Class Website edu/classes/bioc461/ In-class and online lecture notes and illustrations Also: objectives, homework problems, sample questions.

4 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 EXAMINATION POLICY: Three hour examinations (300 points total) No cumulative final examination i Examinations must be taken at scheduled times Some extra credit opportunities will be available FINAL GRADES: (see details in course description) Calculated at end of course relative to % of 300 total points Curved - only to improve grades, if warranted GRADE APPEALS: In writing, see course description/syllabus GRADE POSTING: At D2L site, linked to course home page

5 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 How to succeed in this course Attend lectures and study online course material Read course material before each lecture Take your own notes! Keep notes in a loose leaf book Study to master learning objectives Study Hints - See Objectives on class Website Be familiar with study question formats Form Study Groups - teach each other!

6 GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2010 This can be a challenging g course! Interactive Strategies to Achieve Learning Objectives Lecture Notes Text Web resources Illustrations: tell the story Learning Objectives Study Questions Good Study Habits Lead to Understanding Course Content!

7 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc. Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease August 25, 2008

8 [Fig.4-1] A Nucleic Acid is a Polymer Consist of 3 kinds of components: 1. 5-carbon sugars 2. Phosphates 3. Bases (N-ring structures)

9 [Fig 4.2] 5-Carbon Sugars RNA DNA

10 [Fig 4.4] Bases in Nucleic Acids * 6 A * 2 G * 1 6 * C U T (RNA) (DNA)

11 [Fig 4.4] Bases in Nucleic Acids Dotted blue lines show hydrogen- bonding groups in DNA (RNA) A 2 G C U T (RNA) (DNA)

12 Nucleoside = Sugar+Base Nucleotide = Sugar+Base+Phosphate A G 5 3 A ribonucleotide A deoxyribonucleotide

13 One letter code Three letter code

14 Nucleotides: Generic Names N = any base NMP NDP NTP dnmp dndp dntp one P Two P Three P Example: AMP ADP ATP M = mono D = di T = tri

15 DRAW THE CHEMICAL STRUCTURE [exercise to do for homework do it like a test; look up answers AFTER drawing the structures] damp CMP dtmp

16 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc. Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease

17 [Fig.25.1] Nucleotide Biosynthesis Two major paths are active and important 1) Reuse base PPi * 2Pi* *** Pyrophosphatase enzyme drives reactions and pathways*** etc 2) Make base from simple molecules PPi * 2Pi

18 De novo Pyrimidine Nucleotide Biosynthesis (for RNA and DNA) (CO 2 ) [Fig.25-2] 1. Make the ring, then the nucleotide (UMP) 2. Combine ring with PRPP Many Steps UMP PPi * 2Pi

19 CTP is made from UTP + P + P UMP UDP NH 2 transfer For RNA [Berg, 6 th Ed. P. 713]

20 Leflunomide: A drug for rheumatoid arthritis, cancer [from Jonnie Shackman, 462b Honors project] Rheumatoid arthritis, cancer characterized by excessive RNA synthesis. Carbamoyl phosphate + Asp UMP RNA Uridine Dihydroorotate X Orotate Leflunomide blocks de novo synthesis of UMP In animal models, the effects of leflunomide can be reversed by adding uridine. [cells are rescued by a pyrimidine nucleoside salvage pathway]

21 Regulation of pyrimidine biosynthesis: End product (CTP) feedback inhibition of ATCase (aspartate transcarbamoylase)

22 de novo Purine Nucleotide Biosynthesis (for RNA and DNA) Many Steps The purine ring is built on a ribosephosphate structure [Fig.25.5]

23 Purine Biosynthesis: Committed step * * 2 Pi ** [p.715]

24 IMP: 1 st nucleotide made with a complete purine ring (IMP)

25 AMP and GMP from IMP P-ribosylamine Many Steps 6 IMP 2 2 [Fig.25.8] Aspartate and Glutamine are NH 2 donors

26 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc. Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease

27 AMP and GMP from IMP P-ribosylamine Many Steps 6 IMP 2 2 [Fig.25.8] Aspartate and Glutamine are NH 2 donors

28 Regulation of Purine Biosynthesis: End product feedback complex, reciprocal inhibitions Requires GTP Requires ATP [Fig.25.15]

29 Nucleotide interconversions: To change # of phosphates by successive ATP-driven phosphorylations (catalyzed by kinase enzymes) NMP NDP NTP for RNA dnmp dndp dntp for DNA ATP ADP ATP ADP NMP NDP NTP (1P) (2P) (3P) Some require ATP, some use other nucleotides (NDPs or NTPs) with exchangeable high energy phosphates. Text: p. 713

30 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc.

31 Purine nucleotide salvage pathways GMP insoluble, forms crystals Purine analog to treat gout

32 Hypoxanthine-guanine PRPP transferase(hgrt) [Guanine (Hypoxanthine)+ PRPP GMP] =O in Hypoxanthine 2Pi

33 BASE NUCLEOTIDE PURINE SALVAGE: Phosphoribosyl transferase enzymes (A, G) Base + PRPP (A,G)MP PPi 2Pi 1. Adenine PRPP transferase 2. Hypoxanthine, guanine PRPP transferase [The base in IMP] Diseases: gout and Lesch - Nyhan HGPRT Salvage **Uric acid PRPP Purine de novo Synthesis

34 GOUT AND LESCH-NYHAN SYNDROME Lesch-Nyhan syndrome: - have excessive hyperuricemia (as in gout) - leads to self-mutilation Urate crystals appearing in a diaper often found in synovial fluid of joints

35 Patient with Lesch-Nyhan: Hands bandaged to prevent self-mutilation. Removal of bandages causes patient to become violent.

36 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc.

37 Ribonucleotide reductase: The enzyme that synthesizes deoxyribonucleotides for DNA synthesis - a possible target for antimalarial drugs. [see Biochem 499 Student Project by Hillary Saxon] MALARIA: A target for drugs?

38 NDPs Not one step + P in one step dntps

39 Ribonucleotide Reductase Reaction: NDP s dndp s CDP All 4 NDP s + P All 4 dndp s (A,G,U,C) -P AMP, GMP, UMP CTP

40 Ribonucleotide Reductase: subunits and active site Tyrosine free radical generates X. free radical at active site [NDP substrate binds]

41 Ribonucleotide Reductase: Two kinds of regulatory sites (substrate [NDP] binding) (overall activity) There are 3 different nucleotide binding sites per R1 subunit Regulation maintains balance of cell s dntps

42 Overall activity substrate Specificity R1 subunit dimer

43 Ribonucleotide Reductase: (reaction velocity) (which substrate binds?)

44 dtmp is made from dump Must regenerate CH 2 -THFA ** Thymidylate synthase [Fig 25.12]

45 For DNA: Biosynthetic paths to dtmp,the first thymine-containing nucleotide CTP * UMP 2Pi PPi * ** * 2 more high energy P s for dtmp to dttp

46 CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES Learning objectives: Know the structures and nomenclature of the building blocks of nucleic acids Purine and pyrimidine bases Nucleosides, nucleotides Ribonucleotides and deoxyribonucleotides Know how nucleotides can be interconverted between mono-, di- and trinucleotide forms Know some key reactions in biosynthesis of the triphosphate forms of the four ribonucleotides(for RNA) and the four deoxyribonucleotides (for DNA) Know the importance of nucleotide salvage pathways and the basis of gout and Lesch-Nyhan diease Know why/how nucleotide analogs and inhibitors of thymidylate synthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc.

47 Thymidylate Synthase and DHFR: Targets of drug therapy - cancer and other diseases [Fig.25.13] * Suicide inhibitor (DHFA) Competitive inhibitors (1-C donor to THFA) (THFA) *

48 Dihydrofolate Reductase (DHFR) Methotrexate also binds here

49 Competitive Inhibitors of Dihydrofolate Reductase (DHFR) Folate ananlogs as therapeutic agents Drug Trimethoprim Indications Antibacterial Antifungal Pyrimethamine Antiprotozoal Methotrexate Antineoplastic Antipsoriatic Anti-inflammatory Immunosuppressive

50 Sensitivity of cancers to methotrexate Sensitive Moderately sensitive Not sensitive Acute lymphocytic Head and neck cancer leukemia Acute myelocytic leukemia Burkitts lymphoma Breast cancer Colon cancer Choriocarcinoma Bladder cancer Renal cell cancer Diffuse lymphoma

51 Methotrexate resistance can develop in cancer cells. Dihydrofolate reductase genes (yellow) are amplified (increased in numbers) in cancer cells treated with methotrexate. This allows cancer cells to overproduce dihydrofolate reductase and continue to grow in the presence of methotrexate.

52 * The many roles of folic acid * * * *

53 Actinic keratoses: Develop after too much exposure to the sun 5-Fluorouracil Drug Therapy: topical application 1 week of therapy: Actinic keratosis lesions respond to drug, show inflammatory response. [itchy, painful] 3 weeks of therapy: As abnormal cells are eradicated, skin becomes redder, crusts and peels. [More itchy, painful] 2 weeks after therapy: Destroyed abnormal cells are replaced by healthy cells, forming normal looking skin. End of process much like a peeling sunburn. [mostly itchy]

54 Suicide inhibitor of Thymidylate synthase: 5-fluorodeoxyuracil (5-F-dU), a thymine analog 5-FdU Add deoxyribose-5-p by a salvage pathway using PRPP (similar to purine salvage) Dead end complex: Fluorine can t leave complex [Fig.25.14]

55 Drugs: Anticancer Antiviral Effects on DNA synthesis 1. Thymidylate synthase is target Suicide inhibition 2. Dihydrofolate reductase is target Competitive inhibition 3. Nucleotide analogs AZT: AIDS, Antibreast cancer ddc: Antiviral Stop DNA or RNA synthesis directly (polymerases can t use these. This will be discussed in more detail later in course, starting with Chapter 5)

56 SUMMARY: Main themes of nucleotide metabolism RNA nucleotides (NTPs) DNA nucleotides (dntps) Interconversions of nucleotides Targets of drug chemotherapy

57 SUMMARY Synthesis of RNA nucleotides (NTPs) Purines (A + G) (AMP, GMP) made de novo as purine nucleotides, then phosphorylated to ATP and GTP. Pyrimidines (U + C) UMP to UTP by phosphorylation, then add amino group to get CTP.

58 SUMMARY Synthesis of DNA nucleotides (dntps) da(g,c,u)dps Made from NDPs (ADP, GDP, CDP, UDP) by ribonucleotide reductase as dndps + P dntps dtmp dttp +P +P from dump by thymidylate synthase (first dudp to dutp to get dump)

59 SUMMARY INTERCONVERSIONS OF NUCLEOTIDES: High energy Phosphate exchanges. PURINE SALVAGE AND DISEASES: Uric acid and Gout, Lesch-Nyhan CHEMOTHERAPY: Inhibit specific enzymes of nucleotide 5-FU Methotrexate AZT other nucleotide analogs metabolism. Ultimately prevent cell or virus proliferation by inhibiting RNA or DNA synthesis.