general nucleic acid biochemistry 461, fall...

GENERAL NUCLEIC ACID BIOCHEMISTRY 461, FALL 2006

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, Education Bldg Rm 211• Lecture materials available on web as notes, illustrations, etc.• Textbook, some additional reading.


INSTRUCTOR:

• Dr. Don P. Bourque• Office Hours: [After lectures and by appointment]• Exam Reviews [TBA]

TEACHING ASSISTANTS:

• Jennifer Bethke• Isabel Loftin• Office Hours: [see schedule, TBA]• Reviews: [TBA]

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TEXTBOOK:

• Biochemistry, J.M. Berg, J.L. Tymoczko, L. Stryer, 6th Edition, W.H. Freeman (2006)

CLASS MATERIALS: All available at Class Website

• Lecture notes, illustrations, objectives, homework problems, sample examinations

• http://www.biochem.arizona.edu/classes/bioc461/


EXAMINATION POLICY:

• Three hour examinations (300 points total)• Six (6) quizzes (100 pts total)• Ungraded homework exercises• No cumulative final examination• Examinations must be taken at scheduled times• No make-up exams will be given

FINAL GRADES: (see details in course description)

• Calculated at end of course relative to % of 400 total points• Curved - only to improve grades, if warranted

GRADE APPEALS: In writing, see course description/syllabus

GRADE POSTING: At WebCT site, linked to course home page


How to succeed in this course

! Attend the lectures

! Read lecture 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 sample exam question formats

! Form Study Groups - teach each other!


This can be a challenging course!

Interactive Strategies to Achieve Learning Objectives

Lecture Notes IllustrationsTextWeb resources

Learning Objectives

HomeworksSample Exam Questions

Good Study Habits Lead to Understanding Course Content!

CHAPTER 25: BIOSYNTHESIS OF NUCLEOTIDES

Learning objectives:

• Know the structures and nomenclature of the building blocks of nucleic acids

• 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- andtrinucleotide forms

• Know why nucleotide analogs and inhibitors of thymidylatesynthase are used in drug therapy for diseases such as cancer, AIDS, herpes, etc.

Purine and pyrimidine basesNucleosides, nucleotidesRibonucleotides and deoxyribonucleotides

August 22, 2006

[Fig.4-1] A Nucleic Acid is a Polymer

Consists of:

1. 5-carbon sugars

2. Phosphate

3. Base (N-ring structure)

[Fig 4.2] 5-Carbon Sugars

RNA

DNA

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Produced as a product of the Pentose Phosphate Pathway

[Fig 4.4] Bases in Nucleic Acids

(RNA) (DNA)

167

9

16

6

2

2 22

4 4 4 5

7

A G

C U T

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lines = H bonding in DNA double strand

Nucleoside = Sugar+Base

Nucleotide = Sugar+Base+Phosphate

A ribonucleoside A deoxyribonucleoside

(A deoxynucleotide)

5’3’

A G

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tide

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One letter code

Three letter code

Nucleotides: Generic Names

NMP NDP NTP

dNMP dNDP dNTP

one P Two P Three P

AMP ADP ATP

Example

dAMP CMP dTMP

DRAW THE CHEMICAL STRUCTURE

[exercise to do for homework – do it like a test;

look up answers AFTER drawing the structure]

Nucleotide BiosynthesisTwo major paths are active and important

PPi 2Pi*

PPi 2Pi

*** Pyrophosphatase enzyme drives reactions and pathways***

*

*

Reuse

Makefromsimplemolecules

[Fig.25.1]

PyrimidineNucleotide

Biosynthesis(for RNA and

DNA)

Many Steps

[Fig.25-2](CO2)

2. Combine ring with PRPP

PPi 2Pi

1. Make the ring, then UMP

*UMP

CTP is made from UTP

UMP

UDP

NH2 transfer

For RNA

[Berg, 6th Ed. P. 713]

+ P

+ P

Leflunomide: A drug for rheumatoid arthritis[from Jonnie Shackman, 462b Honors project]

Carbamoyl aspartate

Dihydroorotate Orotate

UMP Uridine

Leflunomide blocks de novo synthesis of UMP

RNA

X

In animal models, the effects of leflunomide can be reversed by adding uridine.[cells are rescued by a pyrimidine nucleoside salvage pathway]

Rheumatoid arthritis is characterized by excessive RNA synthesis.

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

(aspartate transcarbamoylase)

Purine biosynthesis by de novo pathway

Many Steps

The purine ring is built on the ribose-phosphate moiety

[Fig.25.5]

Purine Biosynthesis: Committed step

*

2 Pi*

[p.715]

IMP: 1st nucleotide made with a complete purine ring

(IMP)

AMP and GMP from IMP

IMP

Aspartate and Glutamine are NH2 donors

6

6

2

P-ribosylamine

Many Steps

[Fig.25.8]

Regulation of Purine Biosynthesis: End product feedback

Requires GTP

Requires ATP

[Fig.25.15]

Nucleotide interconversions: To change # of phosphatesby successive ATP-driven phosphorylations

(catalyzed by kinase enzymes)

ATP ADP ATP ADP

NMP NDP NTP

(1P) (2P) (3P)

NMP NDP NTP for RNAdNMP dNDP dNTP for DNA

Some require ATP, some use other nucleotides (NDPs or NTPs) with exchangeable high energy phosphates.Text: p. 713

BASE NUCLEOTIDE

PURINE SALVAGE: Phosphoribosyl transferase enzymes

(A, G) Base + PRPP (A,G)MP

PPi 2Pi

1. Adenine PRPP transferase

2. Hypoxanthine, guanine[The base in IMP]

Diseases: Lesch - Nyhan

HGPRTSalvagePRPPPurine SynthesisUric acid

*

GOUT AND LESCH-NYHAN SYNDROME

Lesch-Nyhan syndrome = excessive hyperuricemia; leads to self-mutilation

Urate crystals appearing in a diaper – often found in synovial fluid of joints

Patient with Lesch-Nyhan:

Hands bandaged to prevent self-mutilation. Removal of bandages causes patient to become violent

MALARIA: A target for drugs?

Ribonucleotide reductase: The enzyme that synthesizes deoxyribonucleotides for DNA synthesis - a possible target forantimalarial drugs.

[Biochem 499 Student Project by Hillary Saxon]

All 4 NDP’s

Ribonucleotide Reductase Reaction:NDP’s dNDP’s

All 4 dNDP’s(A,G,U,C)

AMP, GMP, UMP

CDP

CTP

+ P

- P

Ribonucleotide Reductase subunits and active site

Tyrosine free radicalgenerates X. freeradical at active site

Ribonucleotide Reductase: Two kinds of regulatory sites

3 different nucleotidebinding sitesper R1 subunit

(substrate [NDP]

binding)

Maintains balance of deoxyNTPs for the cell

Ribonucleotide Reductase:(reaction velocity)

(which substrate binds?)

dTMP is made from dUMP

Thymidylate synthase

Must regenerate CH2-THFA

[Fig 25.12]

CTP

*

*

UMP PPi

For DNA: Biosynthetic paths to dTMP,the first thymine-containing nucleotide

2Pi

2 more high energy P’s for dTMP to dTTP

* **

Thymidylate Synthase and DHFR: Targets of cancer drug therapy

(1-C donor

to THFA)

[Fig.25.13]

Competitiveinhibitors

*

*

Competitive Inhibitors of Dihydrofolate Reductase (DHFR) as therapeutic agents

Drug Indications

Trimethoprim AntibacterialAntifungal

Pyrimethamine Antiprotozoal

Methotrexate AntineoplasticAntipsoriaticAnti-inflammatoryImmunosuppressive

Sensitivity of cancers to methotrexate

Sensitive Moderately sensitive Not sensitive

Acute lymphocytic Head and neck cancer Acute myelocyticleukemia leukemia

Burkitts lymphoma Breast cancer Colon cancer

Choriocarcinoma Bladder cancer Renal cell cancer

Diffuse lymphoma

Dihydrofolatereductase genes (yellow) are amplified (increased in numbers) in cancer cells treated with methotrexate.

Actinic keratoses: Develop after too much exposure to the sun

Before therapy: Actinic keratosis lesions are visible

During therapy: As abnormal cells are eradicated, skin becomes redder, crusts and peels.

After therapy: Destroyed abnormal cells are replaced by healthy cells, forming normal looking skin

5-Fluorouracil Drug Therapy: topical application

Thymidylate Synthase and DHFR: Targets of cancer drug therapy

[Fig.25.13]

*

Suicide inhibitor of Thymidylate synthase:

5-fluorodeoxyuracil (5-F-dU), a nucleotide analog

5-FU

Ribose-5-P (a salvage pathway)Dead end complex

[Fig.25.14]

Drugs: Anticancer

AntiviralEffects on DNA synthesis

1. Thymidylate synthaseis target Suicide inhibition

2. Dihydrofolatereductase is target Competitive inhibition

3. Nucleotide analogsAZT: AIDS, AntibreastcancerddC: Antiviral

Stop DNA or RNA synthesis directly (polymerase can’t use these)

SUMMARY: Main themes of nucleotide biosynthesis

RNA nucleotides (NTPs)

DNA nucleotides (dNTPs)

Interconversions of nucleotides

Targets of drug chemotherapy

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.

SUMMARY

Synthesis of DNA nucleotides (dNTPs)

dA, dG, dC (dU) Made from NDPs (ADP, GDP, CDP, UDP)

by ribonucleotide reductase as dNDPs

dNTPs

dTMP from dUMP by thymidylate synthase

(first dUDP to dUTP to get dUMP)

dTTP

+ P

+P+P

SUMMARY

INTERCONVERSIONS OF NUCLEOTIDES: High energy Phosphate exchanges.

PURINE SALVAGE AND DISEASES: Gout, Lesch Nyhan

CHEMOTHERAPY: Inhibit specific enzymes of nucleotide

metabolism. Ultimately prevent cell or

virus proliferation by inhibiting RNA

or DNA synthesis.

5-FU MethotrexateAZTother nucleotide analogs

general nucleic acid biochemistry 461, fall...

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