chemistry of nucleic acid (2)-doc viliran 06/11/09

8/14/2019 Chemistry of Nucleic Acid (2)-doc viliran 06/11/09

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Nucleic acid ChemistryNucleic acid Chemistry

Dolores V. Viliran, M.D.Dolores V. Viliran, M.D.

FEU-NRMF, Institute of MedicineFEU-NRMF, Institute of Medicine

Department of BiochemistryDepartment of Biochemistry


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Function of Nucleic AcidsFunction of Nucleic Acids Nucleic acid= an informational molecule

Two kinds:

DNA- deoxyribonucleic acid

RNA- ribonucleic acid

DNA = blueprint of living organisms Contains genetic information for the synthesis of

proteins

RNA= machinery for the expression of the

coded information in DNA to proteins

mRNA- Carrier of genetic information encoded in

the DNA


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Two Kinds of Nucleic acidTwo Kinds of Nucleic acid

1. DNA = deoxyribonucleic acid

2. RNA = ribonucleic acid

Primary function ofDNA:Storage of genetic material

Central Dogma of LIFE : flow of genetic material

DNA mRNA protein

Replication transcription translation


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PRESENT CONCEPT OF THEPRESENT CONCEPT OF THE

CENTRAL DOGMACENTRAL DOGMADNA RNA PROTEIN

= specific transfer of information from RNAto RNA has been observed in viralsystems

= specific transfer of information from RNAto DNA has been observed in viralsystems and tumor.


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How does DNA and RNA performHow does DNA and RNA perform

these various functions?these various functions?

Structure is intimately related to function

Change the structure and the function may

be lost

Thus we need to know the structure in

order to understand the function


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Covalent Structure of Nucleic acidsCovalent Structure of Nucleic acidsGenetic information is encoded in DNA and carried byGenetic information is encoded in DNA and carried by

mRNA as sequence or a chain of nucleotidesmRNA as sequence or a chain of nucleotides

Composed of polymers of nucleoside

monophosphates(nucleotide-nucleotide-nucleotide)

1. Monomeric units consist of: NUCLEOTIDE

Base-sugar-phosphate (bsp)a. Nitrogenous base which may be a purine or a pyrimidine

b. A pentose sugar, ribose or deoxyribose, in a furanose ring

form

c. A phosphate group esterified to the sugar

Nucleoside- coupling of a base and a sugar

Nucleotide- when a nucleoside becomes phosphorylated


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Nitrogenous base

Purines = adenine(A), Guanine (G)

Pyrimidines = cytosine (C),uracil (U), Thymine(T)

Sugar

DNA = deoxyribose

RNA = ribose

Sugar hold the base on one side and the

phosphate on the other side.

Thus , sugar hold the components of the

nucleotide together


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Nucleotides in DNA

1) ADENINE-2-deoxyRIBOSE-PHOSPHATE

2) GUANINE-2-deoxyRIBOSE-PHOSPHATE

3) CYTOSINE-2-deoxyRIBOSE-PHOSPHATE

4) THYMINE-2-deoxyRIBOSE-PHOSPHATE

Nucleotides in RNA

1) ADENINE-RIBOSE-PHOSPHATE

2) GUANINE-RIBOSE-PHOSPHATE

3) CYTOSINE-RIBOSE-PHOSPHATE

4) URACIL-RIBOSE-PHOSPHATE


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Nucleotides in DNA

ADENINE-2-deoxyRIBOSE-PHOSPHATE

GUANINE-2-deoxyRIBOSE-PHOSPHATE

CYTOSINE-2-deoxyRIBOSE-PHOSPHATE

THYMINE-2-deoxyRIBOSE-PHOSPHATE

Nucleotides in RNA ADENINE-RIBOSE-PHOSPHATE

GUANINE-RIBOSE-PHOSPHATE

CYTOSINE-RIBOSE-PHOSPHATE

URACIL-RIBOSE-PHOSPHATE


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Chemical structure of nitrogenous basesChemical structure of nitrogenous bases

PURINES


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Chemical structure of nitrogenous basesChemical structure of nitrogenous bases

PYRIMIDINES


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SUBSTITUTIONSSUBSTITUTIONSOXYGEN,AMINO GROUPS,METHYL GROUPSOXYGEN,AMINO GROUPS,METHYL GROUPS

PURINES ADENINE = 6-aminopurine

GUANINE = 2-amino,6-oxypurine

PYRIMIDINES CYTOSINE = 2-oxy,4-amino pyrimidine

URACIL = 2,4-dioxypyrimidine

THYMINE = 2,4-dioxy,5-methylpyrimidine


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NucleosidesNucleosidesPurines = N9-C1 glycosidic bondPurines = N9-C1 glycosidic bond

Pyrimidines = N1-C1 glycosidic bondPyrimidines = N1-C1 glycosidic bond

Addition of pentose sugar to a base

produces a nucleoside.

Adenine Adenosine

Guanine Guanosine

Cytosine Cytidine

Thymine ThymidineUracil Uridine


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NUCLEOSIDENUCLEOSIDE


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NUCLEOTIDESNUCLEOTIDES

Nucleotides can have one,two or three

phosphates

Nucleoside Nucleotide

Adenosine Adenosine mono/di/tri phosphate

Guanosine Guanosine mono/di/tri phosphate

Cytidine Cytidine mono/di/tri phosphateUridine Uridine mono/di/tri phosphate

Thymidine Thymidine mono/di/tri phosphate

AMP/ADP/ATP;GMP/GDP/GTP;CMP/CDP/CTP;UMP/UDP/UTP TMP/TDP/TTP


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Covalent Structure of Nucleic acidsCovalent Structure of Nucleic acids

A. Nucleotides- are linked together by

phosphodiester bonds between the 3 hydroxyl

on the sugar of one nucleotide and the 5-

phosphate on the sugar of another nucleotide1. Trinucleotides- addition of another nucleotide at the

3 hydroxyl of the sugar

2. These compounds have polarity of direction- 5 3

3. Characteristically nucleotides are acidic by virtue oftheir phosphate groups


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NUCLEOTIDE POLYMERSNUCLEOTIDE POLYMERS

(NUCLEIC ACID)(NUCLEIC ACID)


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Nomenclature of NUCLEIC ACIDSNomenclature of NUCLEIC ACIDS

A. Polynucleotides

B. Polyribonucleotides polynucleotides containingribose as opposed to de-oxyribose

3. Naturally occuring are called RNA

4. Bases found in RNAs (w/ a few exception) guanine,cytosine, adenine, and uracil

C. Differences between the Primary Structure of DNA andRNA

6. DNA has a 2-deoxyribose as its sugar moiety ratherthan ribose

7. DNA has one different base-thymine (5-methyl-uracil)

8. RNA has one different base-uracil


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Ways of Presenting the structure ofWays of Presenting the structure of

a Polynucleotide (abbreviated form)a Polynucleotide (abbreviated form)

A. 5 p-T-A-C-G-G-G-C-G-A-T-T-T-G-G-GOH

B. 5 pTpApCpGpGpGpCpGpApTpTpTpGpGOH


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DNA THE GENETIC MATERIALDNA THE GENETIC MATERIAL

A. BASIC STRUCTURAL CHARACTERISTICS:

A double Helical Structure as proposed by Watson and

Crick (1953)

3. TWO ANTI PARALLEL POLYDEOXY RIBONUCLEOTIDE

CHAINS Wound around each other with the bases inside of the

helix sugar and PO4 outside

4. STACKING OF THE HYDROHOBIC RINGS Result when the

planes of the bases are perpendicular to the axis of the helix

5. HYDROGEN Bonding between one bases on one stand pairingw/ another base on the anti-parallel strand


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Chargaffs rule: content of A = content of T

content of G = content of C

4. The helix is 2 nm in diameter= bases are separated by 3.4 A along the helixaxis, and each base is rotated 36 in relation tothe previous base.

=the helical structure repeats at intervals of 34 A(every 10 base pairs)

=In solution, the helical structure of DNA probablydoes not repeat every 10-base pairs but nearerto every 11 with the bases inclined 20 from the

horizontalGENETIC INFORMATION IS STORED AS THE

SEQUENCE OF BASES ALONG THE CHAIN


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Secondary structure of DNA: TheSecondary structure of DNA: The

Double HelixDouble Helix


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Right- and left-handed HelicesRight- and left-handed Helices


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A, B, Z forms of DNAA, B, Z forms of DNA


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T ti St t f DNA


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Tertiary Structure of DNATertiary Structure of DNA

SupercoilingSupercoiling

Supercoiling in prokaryotic DNA

Positive supercoils

Negative supercoils

Supercoiling in Eukaryotic DNA

Formation of chromatin

electrostatic attraction between the negatively

charged phosphate groups on the DNA and thepositively charged groups on the protein


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Supercoiling in prokaryotic DNASupercoiling in prokaryotic DNA


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Supercoiling in Eukaryotic DNASupercoiling in Eukaryotic DNA


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Denaturation of DNADenaturation of DNA

The double helix is disrupted during DNA

replication,transcription,repair and

recombination

Therefore the forces that hold the two

strands together are adequate for

providing stability and yet weak enough to

allow easy strand separation


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DISSOCIATION AND REASSOCIATION OF THEDISSOCIATION AND REASSOCIATION OF THE

DOUBLE HELICAL CHAINS OF DNADOUBLE HELICAL CHAINS OF DNA

Double helical chains of DNA have a remarkable ability todissociate from one another and to reassociate again.This behavior is essential to the processes ofREPLICATION and TRANSCRIPTION

DENATURATION rupture of the hydrogen bondsbetween the bases, resulting from increasing temperatureor the alteration of the H+ concentration, which causes thetwo strands to come apart.

3. Increasing pH deprotonates ring nitrogens of guanine

and thymine; decreasing pH protonates ring nitrogens ofadenine and cytosine

4. Increasing acidity can cause rupture of purine glycosidicbonds, and at high temperatures phosphodiester bondsmay be broken.


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Alkali method of choice for DNA denaturation

3. Increasing temperature the double helical

chain dissociates at a definite temperatureknown as Melting temperature (Tm)

c. Tm is the temp. at which 50% of the double

helix is unwound

d. Melting causes unstacking of the base pairswhich results in increased absorbance

(hyperchromic effect)

e. Tm is strongly influenced by the basecomposition of the DNA


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DNA rich GC pairs has a higher Tm thanDNA with a higher proportion of AT pairs

Mammalian DNA, which is about 40%GC pairs has a Tm of about 87oC

B. RENATURATION (Annealing)

If the temperature of melted(dissociated)duplex DNA is rapidlyreduced, the original double helicalstructure does not reform (anneal)

If, however, the temp. is held at a valueof about 20oC to 25oC below the Tm, theoriginal double helical structure reforms.


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Denaturation-RenaturationDenaturation-Renaturation


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S fS t f l ti l ti hi


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T f DNA t t


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Types of DNA structureTypes of DNA structure1. Size of DNA is highly variable

a. DNA size can be expressed as number of

base pairs, molecular mass, the length of the

strands and actual mass of DNA

DNA of molecular weight 1 x 106

contains 1500 bp and is 0.5 nm long

b. The amount of DNA per cell increases as the

complexity of the cellular function increases

- the DNA in the cell is packaged as 46

chromatin fibers

* metaphase- condensed state the largest chromosome

is about 10m if stretched out is about 8 cm long


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Types of DNA structureTypes of DNA structure

1. Size of DNA is highly variable

2. Techniques of determining DNA size

a. Equilibrium centrifugation:CesiumChloride

b. Electron microscopy

c. Electrophoresis


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1. Size of DNA is highly variable

2. Techniques of determining DNA size

3. DNA maybe linear or circulara. Double-stranded circles

b. single-stranded DNA

c. Circular DNA is a superhelix


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T f DNA t tT f DNA t t


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Alternative DNA conformations

a. DNA bending = DNA sequences with

runs of 4 to 6 Adenine bases phased by

10-bp spacers produce bend

conformations

= important in the interaction between

DNA sequences and proteins that

catalyze replication, transcription andsite-specific recombination

T f DNA t tT f DNA t t


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Alternative DNA conformations

Cruciform DNA= dos DNA (defined ordered sequence DNA)

- inverted repeat ( palindromes)

-mirror repeats

- Direct repeats

= present within noncoding DNA regions

= inverted repeats may function as a molecular switchesfor replication and transcription


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Triple-Stranded DNATriple-Stranded DNA

Formed in DNA regions with

continuous string of of purine

bases, that is homopurine-

homopyrimidine regions

Generated by the hydrogenbonding of a third strand into

the major groove of B-DNA

The third strand forms

hydrogen bonds with anothersurface of the double helix

thru Hoogsteen pairs

Limited to only four triplet

bases TAT,CGC,GGC,AAT


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Triple-stranded DNATriple-stranded DNA

Polypurine-polypyrimidine

regions have potential

biological roles: transcription

control,initiation of

replication, replicationterminators,enhancers of

stability of

telomeres,initiators of genetic

recombination


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Four Stranded DNAFour Stranded DNA


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Four-Stranded DNAFour-Stranded DNA

(Quadruplex)(Quadruplex) May form during DNA

recombination

Contains repeated

motifs high in guaninecontent (G-quartet

DNA)

Form at telomeres

Sli d DNASlipped DNA


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Slipped DNASlipped DNA Slipped,mispaired DNA

(SMP-DNA) Formation involves the

unwinding of the double

helix and realignment and

subsequent pairing of onecopy of the direct repeat

with an adjacent copy on

the other strand-

generating a single-

stranded loop


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EUKARYOTIC DNA:EUKARYOTIC DNA:

1. DNA is packaged into unit structures calledCHROMOSOMES

2. Combined with proteins called CHROMATIN chromatincontains about equal amounts (by weight) of DNA andprotein.

3. DNA is associated with basic proteins called histonesand with nonhistones chromosomal proteins. Theseare non-covalent associations

d. Histones are small proteins that carry a considerable(+) charge devided into 5 classes H1;H2A;H2B, H3

and H4


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Chromosomal OrganizationChromosomal Organization

If the DNA of all 46 chromosomes were

lined up in the B-DNA conformation it

would be more than 2 meters long

DNA needs to fit within a cell nucleus witha diameter of approximately 5 m

Packaging of DNA

-Beads-on-a-string: DNA association with histones- 10 nm fiber

- 30 nm fiber (solenoid)

Histone OctamerHistone Octamer


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Histone OctamerHistone Octamer

inserted into the nucleosomeinserted into the nucleosome


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CYTOPLASMIC DNA INCYTOPLASMIC DNA IN


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CYTOPLASMIC DNA INCYTOPLASMIC DNA IN

EUKARYOTIC ORGANELLES:EUKARYOTIC ORGANELLES:

Mitochondria and chloroplasts of eukaryotic cells containDNA that differs from nuclear DNA

2. Neither mitochondrial nor chloroplasts DNA isassociated with histones

3. Mitochondrial DNA of animal cells is:d. Double stranded

e. Circular

f. About 15,000 base-pairs in length

3. Mitochondrial Dna sequences code for only about 5% ofthe protein components of mitochondrial structure andfunction. The bulk of information for mitochondrialprotein synthesis is stored in nuclear DNA


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CHEMICAL NATURE OF RNA;CHEMICAL NATURE OF RNA;1. Sugar moiety is ribose rather than 2

deoxyribose of DNA

2. Pyrimidine component is URACILribonucleotide

3. Exists as a single strand

mRNA

Cap Poly-A tail

5Gppp A,C,G,U,G,A,U,G,A,C,U,U,A,G,G,C, pApApApApApA-OH 3


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mRNAmRNA


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mRNAmRNACharacteristics:

2. Most frequently synthesized and withmost rapid turnover among the RNAs

3. Synthesized in transcription

4. Carries the genetic information from theDNA (codons- triplet nucleotide) and isused as the template for proteinsynthesis

5. Single stranded

6. Read and translated 5 to 3

7. Has methylguanosine cap at the 5 end

and a polyadenine tail

tRNA


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tRNACharacteristics:

3. With the lowest molecular weight

4. Synthesized during transcription5. With about 60 different types, with atleast one tRNA for every AA

some amino acids have 2 or more corresponding tRNAs

6. Transfers amino acids from the cytoplasm to the ribosomes

7. adaptor molecule that carries specific amino acid to the site of

protein synthesis. There, if recognizes the genetic code word that

specifies the addition of its amino acid to the growing peptide chain

8. Contains the anti-codon

9. Single stranded but assumes a cloverleaf structure (secondary level)

10. Contains unusual/rare bases like ribothymine, pseudouracil anddihydroxyuracil

11. Amino acid is always attached to the 3 end and the 3 end always

end in CCA sequence

12. Amino acid is attached to the tRNA during the activation stage of

translation

tRNA


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tRNAtRNA

rRNA


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rRNA

Characteristics:

3. Intimately associated with ribosomes4. Most abundant of all RNAs

5. Least characterized among all RNAs

and is synthesized during transcription

rRNArRNA


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rRNArRNA


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METABOLISM OF PURINE AND

PYRIMIDINE NUCLEOTIDES

Biomedical ImportanceBiomedical Importance


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Biomedical Importancep

The biosynthesis of purines and pyrimidines is

stringently regulated and coordinated byfeedback mechanisms that ensure their

production in quantities and at times appropriate

to varying physiologic demand

Genetic diseases of purine metabolism include

gout, Lesch-Nyhan syndrome, adenosine

deaminase deficiency, and purine nucleoside

phosphorylase deficiencyBy contrast, apart from the orotic acidurias, there

are few clinically significant disorders of

pyrimidine catabolism.

P i & P i idi A Di t il N ti lP i & P i idi A Di t il N ti l


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Purines & Pyrimidines Are Dietarily NonessentialPurines & Pyrimidines Are Dietarily Nonessential

Human tissues can synthesize purines and pyrimidines from

amphibolic intermediates Ingested nucleic acids and nucleotides, which therefore are

dietarily nonessential, are degraded in the intestinal tract to

mononucleotides, which may be absorbed or converted to

purine and pyrimidine bases The purine bases are then oxidized to uric acid, which may be

absorbed and excreted in the urine

While little or no dietary purine or pyrimidine is incorporated

into tissue nucleic acids, injected compounds are incorporated The incorporation of injected [3H]thymidine into newly

synthesized DNA thus can be used to measure the rate of

DNA synthesis.

Role of Nucleotides in Biochemical Processes:Role of Nucleotides in Biochemical Processes:


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Role of Nucleotides in Biochemical Processes:Role of Nucleotides in Biochemical Processes:

1. Activated precursors of DNA and RNA

2. Activated intermediates in many biosynthetic reactions

C. UDP GLUCOSE Glycogen synthesis

D. CDP GLYCEROL Phosphoglyceride synthesis

E. s-ADENOSYL METHIONINE methylation reactions

3. Energy SourcesG. ATP universal energy currency

H. GTP translocation of nascent peptide chains onribosomes

- Activation of signal coupling proteins

4. ADENINE NUCLEOTIDES COMPONENTS OF THREECO-ENZYMES: NAD, FAD, AND Co-A

5. METABOLIC REGULATORS

i.e. c-AMP intra-cellular secondary messenger

Biosynthesis of Purine NucleotidesBiosynthesis of Purine Nucleotides


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yy

P i N l tid Bi th iPurine Nucleotide Biosynthesis


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Purine Nucleotide BiosynthesisPurine Nucleotide Biosynthesis

Three processes contribute to purinenucleotide biosynthesis

(1) synthesis from amphibolic intermediates

(synthesis de novo)

(2) phosphoribosylation of purines

(3) phosphorylation of purine nucleosides.

Inosine Monophosphate (IMP) IsInosine Monophosphate (IMP) Is

S th i d f A hib li I t di tS th i d f A hib li I t di t


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Synthesized from Amphibolic IntermediatesSynthesized from Amphibolic Intermediates

C i f IMP t AMP d GMPConversion of IMP to AMP and GMP


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Conversion of IMP to AMP and GMPConversion of IMP to AMP and GMP

"Salvage Reactions" Convert Purines &"Salvage Reactions" Convert Purines &

Th i N l id t M l tidTheir N cleosides to Monon cleotides


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Their Nucleosides to MononucleotidesTheir Nucleosides to Mononucleotides

Reduction of Ribonucleoside DiphosphatesReduction of Ribonucleoside Diphosphates


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p pp p

Forms Deoxyribonucleoside DiphosphatesForms Deoxyribonucleoside Diphosphates

Regulation of the reduction of purine andRegulation of the reduction of purine and

pyrimidine ribonucleotides to their respectivepyrimidine ribonucleotides to their respective


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pyrimidine ribonucleotides to their respectivepyrimidine ribonucleotides to their respective

2'-deoxyribonucleotides2'-deoxyribonucleotides

Biosynthesis of Pyrimidine NucleotidesBiosynthesis of Pyrimidine Nucleotides


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PURINE NUCLEUSPURINE NUCLEUS


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PURINE NUCLEUSU UC USSOURCES:

N-3 and N-9 from the amide nitrogen of Gln C-4, C-5 and N7 from glycine

C-2 form N10 formyl H4 folate

C-8 from N5 N10 methenyl H4

folate

4. C-6 from CO2

5. N-1 from aspartate

Pyrimidine Nucleus

SOURCES:

10. N-1, C-4, C-5 and C-6 from Asp

11. C2 from CO2

12. N3 from the amide N of Gln (glutamine)

FORMATION OF DEOXYRIBONUCLEOTIDES

ADP dADP


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GDP dGDP

UDP dUDP

CDP dCDP

-produces dNTPs for DNA replication

-enhanced during the S phase of cell cycle (prior to celldivision)

-formed from the reduction of the correspondingribonucleosides diphosphates.

-enzyme ribonucleoside reductase

direct reducing of Hydrogen donor: thioredoxin (Sh

containing protein)ultimate hydrogen donor: NADPH

enzyme to re-reduce thioredoxin: thioredoxin reductase

-subject to regulation

FORMATION OF DEOXYRIBONUCLEOTIDESFORMATION OF DEOXYRIBONUCLEOTIDES


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FORMATION OF DEOXYRIBONUCLEOTIDESFORMATION OF DEOXYRIBONUCLEOTIDES

NMP

ATP

ADP

NDP

Ribonucleotide thioredoxin (SH)2 NADP

Reductase

thioredoxin (S-S) NASPH+H

dNDP

ATP

ADP

dNTP

FORMATION OF TMPFORMATION OF TMP


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FORMATION OF TMPFORMATION OF TMP-In the formation of dTMP from dUMP, FH4 is the methyl donor which

simultaneously is oxidized to FH2, this requiring reduction by dihydrofolate reductase.

-This reaction is most sensitive to the inhibitory effect of aminopterin oramethopterin (methotrexate).

-Aminopterin is an analogue of folic acid thus competitively inhibitingdihydrofolate reductase, preventing the formation of FH4, the activeform of the enzyme.

Hence, this drug is used in cancer chemotherapy because of TMPsynthesis, and ultimately DNA replication.

-Trimethoprim (present in Bactrim, Cotrimoxazole) has similarmechanism of action but acts more on bacterial cells, hence usedas antibiotics.

-Trimethoprim is generally used in combination with sulfonamides, apara-aminobenzonic acid (PABA) analogue. PABA is a constituentsof folic acid, hence the presence of sulfonamides will prevent folicacid synthesis in bacteria (but not in man, as folic acid is ingestedpre-formed in man as a vitamin), thus inhibiting TMP synthesis andDNA replication of bacterial leading to cell death

FORMATION OF TMPFORMATION OF TMP


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dUTP dCPM

Hydrolase DeaminasePpi NH3

dUMP

N5 N10 methylene

FH4

Dihydrofolate Thymidylate

Reductase synthetase

FH2 dMTP

SALVAGE PATHWAYS FOR PURINE ANDSALVAGE PATHWAYS FOR PURINE AND


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PYRIMIDINE BASESPYRIMIDINE BASES

-important in cells which cannot carry out the de

novo pathways e.g. due to lack of enzyme PRPP

amidotransferase (rate limiting step in purine

nucleotide biosynthesis) as in red blood cells.

-more economical in terms of high energyphosphates expenditure compared to the de

novo pathway.

-nucleosides, which can enter cells (nucleotides

cannot) are either converted to a nucleotide thru

the action of a kinase or degraded to a base by

nucleoside phosphorylase

ENZYMESENZYMES


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1. Hypoxanthine guanine phosphoribosyl

transferase (HGPRTase) catalyzes the one-step formation of hypoxanthine or guanine to

IMP or GMP respectively

Guanine + PRPP GMP+PPi

Hypoxanthine + PRPP IMP + PPi

Complete deficiency of this enzyme produces

Lesch-Nyhan Syndrome characterized by:1. Hyperuricemia and gout

2. Psychomotor abnormalities

3. Mental retardation

4. Self mutilation

2. Adenine phosphoribosyl transferase (APRTase)

t l th i f d i t AMP


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catalyzes the conversion of adenine to AMP

Adenine + PRPP AMP+PPi

3. Pyrimidine phosphoribosyl transferase catalyzes

the conversion of orotate, uracil and thymine to

their corresponding nucleotides.

Orotate+PRPP OMP+PPiUracil + PRPP UMP+PPi

Thymine + PRPP TMP+PPi


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FORMATION OF TMP

-In the formation of dTMP from dUMP, FH4is the methyl donor which simulatneously

is oxidized to FH2, this requiring reduction bydihy drofolate reductase.

-This reaction is most sensitive to the inhibitory

effect of aminopterin or amethopterin

(methotrexate)

FORMATION OR DEOXYPYRIMIDINEFORMATION OR DEOXYPYRIMIDINE

C O S O S S S


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NUCLEOTIDES FOR DNA SYNTHESISNUCLEOTIDES FOR DNA SYNTHESIS

CDP dCDP dCMP dUMp

dCTP dTMP

DNA dTDP

dTTP

SALVAGE PATHWAYSSALVAGE PATHWAYS


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SALVAGE PATHWAYSSALVAGE PATHWAYS

FREE BASE (PURINES, PYRIMIDINES)RIBOSE-1-P

PRPP PI

PPi nucleosides

NMP ATP

ATP Kinase

NDP

ATP Kinase

NTP


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Degradation of Pyrimidine Bases:

Deaminase reductase


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Deaminase reductaseCytosine Uracil Dihydrouracil

H20 NH4+ NADPH NADPH20

B-Ureidopropionate

B-alanine

CO2+NH3

B-aminoisobutyrate

B-ureidoisobutyrate

deaminase reductase H20

Methyl cytosine thymine dihydrothymine

H20 NH4 NADPH NADP

-Ring opening gives rise to B-

id i t d B id i b t t


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ureidopropionate and B-ureidoisobutyrate

-uracil and cytosine eventually gives rise toB-alanine

-B-aminosobutyrate, the degradation

product of thymine can be used tomeasure DNA turnover

Degradation of Pyrimidine Bases:

Deaminase reductase


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Deaminase reductaseCytosine Uracil Dihydrouracil

H20 NH4+ NADPH NADPH20

B-Ureidopropionate

B-alanine

CO2+NH3

B-aminoisobutyrate

B-ureidoisobutyrate

deaminase reductase H20

Methyl cytosine thymine dihydrothymine

H20 NH4 NADPH NADP

Disorders of Purine CatabolismDisorders of Purine Catabolism


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HYPERURICEMIA1. Gout

2. Lesch-Nyhan syndrome

defect in hypoxanthine-guanine phosphoribosyl

transferase

3. Von Gierke's Disease

hyperuricemia in von Gierke's disease (glucose-6-

phosphatase deficiency) occurs secondary to

enhanced generation of the PRPP precursor ribose 5-phosphate

An associated lactic acidosis elevates the renal

threshold for urate, elevating total body urates.


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HYPOURICEMIA

Hypouricemia and increased excretion of hypoxanthine and xanthine

1. xanthine oxidase deficiency due to a genetic defect or to severe liverdamage. Patients with a severe enzyme deficiency may exhibit

xanthinuria and xanthine lithiasis.

2. Adenosine Deaminase & Purine Nucleoside Phosphorylase Deficiency

Adenosine deaminase deficiencyis associated with an immunodeficiency

disease in which both thymus-derived lymphocytes (T cells) and bone

marrow-derived lymphocytes (B cells) are sparse and dysfunctional

Purine nucleoside phosphorylase deficiency is associated with a severe

deficiency of T cells but apparently normal B cell function

Immune dysfunctions appear to result from accumulation of dGTP anddATP, which inhibit ribonucleotide reductase and thereby deplete cells of

DNA precursors.


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Diagnosis:

-Colchicine trial


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-synovial fluid finding

needle shaped

Negatively birefringent

Wbc count 10,000 ~ 60,000

Neutrophil predominance

-Tophi

Cream colored, firm, chalky, movable Punched-out erosions

Overhanging edge

Treatment

-colchicine-NSAIDs(indomethacin)

-Corticosteroids

-allopurinol (xanthine oxidase inhibitor)

-Probenecid (uricosuric)

Diet therapy:

Purines: low


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Calories: maintain ideal weight

Carbohydrates: highProteins: moderate

Fats: low

Fluids: large amounts

Foods:

High Purine Content

Anchovies Bouillon Brain Broth

Goose Gravy Heart Herring

Kidney Liver Mackerel yeast

Meat extracts & mince mussels portridge

Roe sardines scallops sweetbread

Moderate purine content

Meat & fish except those listed above


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Meat & fish except those listed abovePoultry shellfish asparagus beans

Lentils mushroom peas spinach

Negligible purine content

Bread crackers butter margarinesCake cereal cheese chocolate

Coffee cornbread ice cream milk

Noodles nuts oils olives

Pickles popcorn pudding rice

Soft drinks eggs fruits gelatin

Tea vegetables (except those listed above)

Disease associated with defects ofDisease associated with defects of

nucleotide metabolismnucleotide metabolism


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nucleotide metabolismnucleotide metabolism1. Hyperuricemia and gout

Deposition of tophi in soft tissues and jointsCan be due to inability to regulate the production of purine nucleotides e.g.

overactivity of PRPP synthetase or PRPP amidotransferase

2. Lesch-Nyhan Syndrome

Defeciency of HGPRT ase (enzyme of the salvage pathway)

3. Von-Gierkes disease- the deficiency of glucose-6-phosphataseincreases the levels of glucose-6-P which in turn enhance thepentose phosphate pathway (HMP shunt) increasing intracellularlevels of ribose -1 phosphate. Elevated levels of ribose-1-phosphateincreases cellular production of PRPP with consequentoverproduction of purine nucleotides, thus HYPERURICEMIA

4. Hypouricemia associated with deficiency of xanthine oxidase or purinenucleoside phosphorylase (no degredation of purines to uric acid)can be due to severe liver damage. Can exhibit xanthinuria andxanthine lithiasis if deficiency is severe.

5. Immunodeficiency diseases associated with

adenosine deaminase deficiency


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adenosine deaminase deficiency

-there is dysfunction of T-cells and B-cells

apparently due to the inhibitory effect of dATPwhich accumulates in this disorder dATP is a

strong inhibitor of this ribonucleotide reductase,

an important enzyme in the synthesis of dNTPs

depleting cells of DNA precursors particularly

dCTP.

6. Immunodeficiency diseases associated with

purine nucleoside phosphorylase deficiency.-impaired T-cell function but normal B-cell

Catabolism of Pyrimidines ProducesCatabolism of Pyrimidines Produces

Water Soluble MetabolitesWater Soluble Metabolites


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Water-Soluble MetabolitesWater-Soluble Metabolites

Catabolism of PyrimidinesCatabolism of Pyrimidines


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yy

the end products of pyrimidine catabolismare highly water-soluble: CO2, NH3,

-alanine, and aminoisobutyrate

Excretion of -aminoisobutyrate increasesin leukemia and severe x-ray radiation

exposure due to increased destruction of

DNA.

Disorders associated with defectsDisorders associated with defects

in pyrimidine metabolismin pyrimidine metabolism


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in pyrimidine metabolismin pyrimidine metabolism

1. Orotic aciduria-characterized by high levels of urinary orotic acid, retarded

growth and megaloblastic anemia.

-there is a deficiency of orotate phosphoribosyl transferase,orotate decarboxylase or both which prevents the

formation of UTP or CTP inhibiting the carbamoylphosphate synthetase step or ATCase steprespectively.

-the failure to synthesize pyrimidine nucleotides is also thecause for growth retardation and megaloblastic

anemia.-can be treated by giving uridine (make pyrimidine essentialconstituents of the diet since the de novo pathwaycannot provide adequate amounts of pyrimidinenucleotide synthesis. This will also exert a feedbackinhibition on carbamoyl phosphate synthetase reaction

2. Ornithine Transcarbamoylase Deficiency (OTCD)-Characterized by orotic aciduria


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Characterized by orotic aciduria

-due to accumulation of carbamoyl phosphate

-deficiency of this enzyme prevents the formation ofcitrulline from the reaction between carbamoyl

phosphate and ornithine (part of the urea cycle) hence

producing elevated levels of carbamoyl phosphate.

-elevated levels of carbamoyl phosphate in turn stimulatesthe rate limiting step in the pyrimidine synthesis pathway

producing elevated levels of orotic acid

-manifestation of protein intolerance and hyperammonemia

with hepatic encephalophaty are also apparent becauseof the failure to detoxify ammonia as a consequence of

the deficiency of the enzyme.

INHIBITORS OF PURINE METABOLISMINHIBITORS OF PURINE METABOLISM

(FOR CANCER THERAPY)(FOR CANCER THERAPY)


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(FOR CANCER THERAPY)(FOR CANCER THERAPY)The turnover of nucleic acids in malignant tissues is very

high, thus inhibition in its formation may have atherapeutic effect. Can also affect normal host cellswhich are rapidly dividing like the hematopoieticsystem.

2. 6-mercaptopurine (6MP)-an anti-tumor drug: ananalogue of adenine metabolized to a ribonucleotide bythe APRT salvage pathway thus inhibiting conversionof IMP to GMP or AMP. It also inhibits the rate-limitingstep of the de novo pathway. Simultaneous

administration with allopurinol potentiates its effects as6MP eill not be degraded and therefore will delay itsactivation.

2. Adenosine arabinoside (sugar is replaced

by arabinose) used as an antiviral and


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by arabinose)- used as an antiviral and

anti-tumor agent in man; inhibits DNApolymerase after it has been metabolized

to the triphosphate form.

3. Azaserine-an analogue of Gln, thus,

inhibits the incorporation of N9 and N3 into

the purine ring, inhibits formation of GMP

from IMP, CTP from UTP reactions

requiring GLN.

Inhibitors to pyrimidine metabolismInhibitors to pyrimidine metabolism

for cancer/viral chemotherapyfor cancer/viral chemotherapy


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for cancer/viral chemotherapyfor cancer/viral chemotherapy

1. Aminopterin and methotrexate inhibits dihydrofolatereductase

2. 5-flouduroucil (5-FU)

-an analogue of thymine used in the treatment of solidtumors converted to the monophosphate nucleotideform via the salvage pathway

-eventually converted to the deoxyribonucleotide form andbinds to thymidylate synthetase, thereby inhibiting theformation of TMP.

-as a deoxytriphosphate form, can be incorporated intoRNA and inhibits the formation of mature RNA(important in translation)


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SummarySummary


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y

Ingested nucleic acids are degraded to purines andpyrimidines. New purines and pyrimidines are formed

from amphibolic intermediates and thus are dietarily

nonessential.

Several reactions of IMP biosynthesis require folatederivatives and glutamine. Consequently, antifolate

drugs and glutamine analogs inhibit purine biosynthesis.

Oxidation and amination of IMP forms AMP and GMP,

and subsequent phosphoryl transfer from ATP formsADP and GDP. Further phosphoryl transfer from ATP to

GDP forms GTP. ADP is converted to ATP by oxidative

phosphorylation. Reduction of NDPs forms dNDPs.

SummarySummary


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Hepatic purine nucleotide biosynthesis is stringentlyregulated by the pool size of PRPP and by feedback

inhibition of PRPP-glutamyl amidotransferase by AMP

and GMP.

Coordinated regulation of purine and pyrimidinenucleotide biosynthesis ensures their presence in

proportions appropriate for nucleic acid biosynthesis and

other metabolic needs.

SummarySummary Humans catabolize purines to uric acid (pKa 5.8),


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present as the relatively insoluble acid at acidic pH or

as its more soluble sodium urate salt at a pH nearneutrality. Urate crystals are diagnostic of gout. Other

disorders of purine catabolism include Lesch-Nyhan

syndrome, von Gierke's disease, and hypouricemias.

Since pyrimidine catabolites are water-soluble, their

overproduction does not result in clinical

abnormalities. Excretion of pyrimidine precursors can,

however, result from a deficiency of ornithinetranscarbamoylase because excess carbamoyl

phosphate is available for pyrimidine biosynthesis.

This DNA form is seen physiologic conditionsThis DNA form is seen physiologic conditions

where cell is well hydrated:where cell is well hydrated:


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yy

A. A form B. B form

C. Z form

D. D form


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TRUE regarding salvage pathways ofTRUE regarding salvage pathways of

nucleotide metabolism:nucleotide metabolism:


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nucleotide metabolism:

A. They utilize free bases as substrates fornucleotide biosynthesis

B. They take place only in the mitochondria

C. They derive the ring from amphibolicintermediates

D. They are more efficient than de novo pathway

The enzyme that catalyzes the committedThe enzyme that catalyzes the committed

step in purine de novo pathway:step in purine de novo pathway:


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step pu e de o o pat ayp p p y

A. Xanthine oxidase B. PRPP synthase

C. PRPP glutamyl amido transferase

D. HGPRTase

The enzyme that catalyzes the committedThe enzyme that catalyzes the committed

step in purine de novo pathway:step in purine de novo pathway:


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p p p yp p p y

A. Xanthine oxidase B. PRPP synthase

C. PRPP glutamyl amido transferase

D. HGPRTase

Xanthine Oxidase inhibitorXanthine Oxidase inhibitor


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A. Azaserine B. Sulfonamides

C. Trimethoprim

D. allopurinol

Xanthine Oxidase inhibitorXanthine Oxidase inhibitor


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A. Azaserine B. Sulfonamides

C. Trimethoprim

D. allopurinol

The first purine nucleotide synthesizedThe first purine nucleotide synthesized

during the de novo pathwayduring the de novo pathway


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g p yg p y

A. AMP B. GMP

C. IMP

D. ADP

The first purine nucleotide synthesizedThe first purine nucleotide synthesized

during the de novo pathwayduring the de novo pathway


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g p yg p y

A. AMP B. GMP

C. IMP

D. ADP

Which enzyme participates in both salvageWhich enzyme participates in both salvage

and De novo pathway?and De novo pathway?


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p yp y

A. Adenosine phosphoribosyl transferase B. Xanthine oxidase

C. Phosphoribosyl phosphate synthase

D. Phosphoribosyl phosphate

amidotransferase

Which enzyme participates in both salvageWhich enzyme participates in both salvage

and De novo pathway?and De novo pathway?


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p yp y

A. Adenosine phosphoribosyl transferase B. Xanthine oxidase

C. Phosphoribosyl phosphate synthase

D. Phosphoribosyl phosphate

amidotransferase

The primary product of purineThe primary product of purine

catabolismcatabolism


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A. Xanthine B. Urea

C. Uric acid

D. hypoxanthine

The primary product of purineThe primary product of purine



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A. Xanthine B. Urea

C. Uric acid

D. hypoxanthine

Which molecule is not aWhich molecule is not a

monomer of ribonucleic acids?monomer of ribonucleic acids?


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A. Adenine B. Thymine

C. Cytidine

D. Uridine

Which molecule is not aWhich molecule is not a



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A. Adenine B. Thymine

C. Cytidine

D. Uridine

Storage molecule of geneticStorage molecule of genetic

information:information:


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information:

A. DNA B. RNA

C. Both

D. Neither

Storage molecule of geneticStorage molecule of genetic

information:information:


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A. DNA B. RNA

C. Both

D. Neither

Hyperchromicity effect is observed in:Hyperchromicity effect is observed in:


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A.DNA renaturation B. DNA denaturation

C. dsDNA

D. RNA

Hyperchromicity effect is observed in:Hyperchromicity effect is observed in:


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A.DNA renaturation B. DNA denaturation

C. dsDNA

D. RNA

If the adenine content of a double-helicalIf the adenine content of a double-helicalDNA is 20% of the total bases, the cytosineDNA is 20% of the total bases, the cytosine

content would be:content would be:


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A. 20%

B. 30%

C. 40%

D. 50%

E. 60%

If the adenine content of a double-helicalIf the adenine content of a double-helicalDNA is 20% of the total bases, the cytosineDNA is 20% of the total bases, the cytosine



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20%

30%

40%

50%

E. 60%


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END OF LECTUREEND OF LECTURE

chemistry of nucleic acid (2)-doc viliran 06/11/09

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