NUCLEIC ACIDS AND NUCLEOTIDESLELLY YUNIARTI, S.SI., M.KES

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Why Study DNA?To truly understand genetics, biologists first had to discover the chemical structure of the geneThis would then help them understand how genes control the inherited characteristics of living thingsGene expression is what enables cells of the same organism to take on so many different sizes, shapes and functions (even though just about every cell in an individual contains the same DNA)

1.What organelle is known as the control center of the cell?

2.What structures are found in the nucleus?

3.What are short segments of chromosomes?

4. What are genes/chromosomes composed of?

5. How do genes/chromosomes control the activity of the cell?

Review

nucleus

chromosomes

genes

DNA

produce proteins thatregulate cell functions and become cell structures

ReviewReview

DNA

• Scientists wondered how DNA worked. They knew genes do these critical things:• Carry information from one generation to another• Put information to work to determine an organism’s characteristics • Can be easily copied• Store and transmit genetic information needed for all cell functions• In order to do these things it had to be a special molecule!

DNA Function

• Our knowledge of DNA put to use:• Inheritance/ Genetic Counseling• Cell function/protein synthesis• Embryonic development/gene regulation• Evolution/ phylogenetic relationships• Medicine/genetic diseases• Genetic engineering/ recombinant DNA

Understanding DNA

DNAPackage

• Chromosomes

1. Are like books full of sentences

2. DNA strand twists around and around itself

DNAPackage

• Nucleus

1. Is like a bookcase

2. Inside the cell, where all the chromosomes are stored

• So what would a library full of rows and rows of bookcases represent?

many cells together

which is a tissue

DNAPackage

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DNADNA FunctionsFunctions 1. Storage of genetic information1. Storage of genetic information 2. Self-duplication & inheritance.2. Self-duplication & inheritance. 3. Expression of the genetic message.3. Expression of the genetic message. DNA’s major function is to code DNA’s major function is to code

for proteins.for proteins. Information is encoded in the order of Information is encoded in the order of

the nitrogenous bases.the nitrogenous bases.

I. Bacterial Transformation is I. Bacterial Transformation is Mediated by DNAMediated by DNA

Experiment by Frederick Griffith – 1928Experiment by Frederick Griffith – 1928 Demonstrated first evidence that genes are Demonstrated first evidence that genes are

moleculesmolecules Two different strains of Two different strains of Streptococcus Streptococcus

pneumoniaepneumoniae Non-pathogenic = Avirulent = ROUGH cells (R)Non-pathogenic = Avirulent = ROUGH cells (R) Pathogenic = virulent = SMOOTH (S)Pathogenic = virulent = SMOOTH (S)

Smooth outer covering = capsuleSmooth outer covering = capsule Capsule = slimy, polysaccharideCapsule = slimy, polysaccharide Encapsulated strains escape phagocytosisEncapsulated strains escape phagocytosis

TransformationTransformation Uptake of genetic material from an external Uptake of genetic material from an external

source resulting in the acquisition of new source resulting in the acquisition of new traits (phenotype is changed)traits (phenotype is changed)

Griffith’s expriment was the earliest Griffith’s expriment was the earliest document evidence of transformationdocument evidence of transformation

THE NUCLEIC ACIDS THE NUCLEIC ACIDS (DNA and RNA) (DNA and RNA)

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Are formed by the polymerization of Are formed by the polymerization of a large number of nucleotide units.a large number of nucleotide units.

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NUCLEOTIDESNUCLEOTIDES

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A nucleotide is a compound that A nucleotide is a compound that containscontains a nitrogenous heterocyclic a nitrogenous heterocyclic base (a purine or a pyrimidine)base (a purine or a pyrimidine)

connected to a phosphorylated connected to a phosphorylated pentose sugar units (deoxyribose or pentose sugar units (deoxyribose or ribose)ribose)

The bases in nucleosides The bases in nucleosides and nucleotidesand nucleotides

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The principal purine bases found in the The principal purine bases found in the body are body are adenineadenine (A), and (A), and guanineguanine (G) (G)

Other purine bases include Other purine bases include xanthinexanthine and and hypoxanthinehypoxanthine

   Pyrimidine bases in the body are Pyrimidine bases in the body are cytosinecytosine

(C), (C), uraciluracil (U) and (U) and thyminethymine (T) (T)

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Nucleoside and nucleotide Nucleoside and nucleotide nomenclaturenomenclature

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Both nucleosides and nucleotides Both nucleosides and nucleotides are named after the bases from are named after the bases from which they are derived.which they are derived.

The nucleosides of ribose with The nucleosides of ribose with adenine, guanine, cytosine, thymine adenine, guanine, cytosine, thymine and uracil are called adenosine, and uracil are called adenosine, guanosine, cytidine, thymidine and guanosine, cytidine, thymidine and uridine uridine

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N9

N

NN

NH2

O

OH OH

OHO

OH OH

OH

N1

N

O

NH2

Adenosine Cytidine Guanosine Uridine

O

OH OH

OH

N9

N

NN

O

NH2

O

OH OH

OH

N1

N

O

O

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The corresponding nucleotides are called The corresponding nucleotides are called adenylic acid or adenosine monophosphate adenylic acid or adenosine monophosphate (AMP)(AMP), , guanilyc acid or guanosine guanilyc acid or guanosine monophosphate (GMP)monophosphate (GMP), , cytidilic acid (CMP), cytidilic acid (CMP), uridylic acid (UTP) and thymidic acid (TMP)uridylic acid (UTP) and thymidic acid (TMP)

If the oxyribose is present rather than If the oxyribose is present rather than ribose, the prefix deoxy is used, as in ribose, the prefix deoxy is used, as in deoxyuridine (dU) or deoxyuridylic acid deoxyuridine (dU) or deoxyuridylic acid (dUMP) (dUMP)

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Note: Note: The monophosphorylated form of adenosine The monophosphorylated form of adenosine (adenosine-5'-monophosphate) is written as, (adenosine-5'-monophosphate) is written as, AMP. The di- and tri-phosphorylated forms are AMP. The di- and tri-phosphorylated forms are written as, ADP and ATP, respectively. written as, ADP and ATP, respectively.

AMP dAMP UMP TMP

OP

O

O

ONN

NN

NH2

O

OH OH

OP

O

O

OO

OH H

NN

NN

NH2

OP

O

O

OO

OH OH

N1

NH

O

O

OP

O

O

OO

OH H

N1

NH

O

O

CH3

Linkage of the base and sugar Linkage of the base and sugar unitsunits

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In both nucleIn both nucleosideosides and nucleotides, the s and nucleotides, the purine or pyrimidine base is linked to the purine or pyrimidine base is linked to the pentose sugar by a glycosidic linkage. pentose sugar by a glycosidic linkage.

The C-1 of the pentose (the anomeric The C-1 of the pentose (the anomeric carbon of the D-ribose or 2'-deoxy-D-carbon of the D-ribose or 2'-deoxy-D-ribose) is linked to the N-1 of the ribose) is linked to the N-1 of the pyrimidine ring or N-9 the purine ring.pyrimidine ring or N-9 the purine ring.

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The atoms in the sugar unit are The atoms in the sugar unit are numbered with prime to distinguish the numbered with prime to distinguish the from atoms in the bases. from atoms in the bases.

It is important to indicate the position at It is important to indicate the position at which esterification of the sugar has which esterification of the sugar has occurred e.g.:occurred e.g.:Adenosine 3′-monophosphate or Adenosine 3′-monophosphate or adenosine 5′-monophosphat.adenosine 5′-monophosphat.

Unless otherwise stated, reference to, for Unless otherwise stated, reference to, for example, and adenine nucleotide in the example, and adenine nucleotide in the text implies that is a 5′- ester.text implies that is a 5′- ester.

Bases, nucleosides, and nucleotidesBases, nucleosides, and nucleotides

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NucleosideNucleosideX=ribose X=ribose

or or deoxyribosdeoxyribos

ee

Nucleotide, Nucleotide, WhereWhere

X=Ribose X=Ribose PhosphatePhosphate

AdenineAdenineAA

GuanineGuanineGG

CytosinCytosineeCC

AdenosineAdenosineAA

GuanosineGuanosineGG

CytidineCytidineCC

Adenosine Adenosine monophosphatemonophosphateAMPAMP

Guanosine Guanosine monophosphatemonophosphateGMPGMP

Cytidine Cytidine monophosphatemonophosphateCMPCMP

N9

N

NN

N H 2

X

NN

NN

O

NH 2

H

X

N

N

O

N H 2

X

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Base FormulaBaseX=H

NucleosideX=ribose or deoxyribose

Nucleotide, WhereX=Ribose Phosphate

UracilU

ThymineT

UridineU

ThymidineT

Uridine monophosphateUMP

Thymidine monophosphateTMP

N

N

O

O

H

X

N

N

O

O

CH3H

dX

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N9

N

NN

NH 2

O

OH OH

OH

syn- Adenosine anti -Adenosine

N9

N

N N

NH 2

O

OH OH

OH

• Nucleosides are found in the cell primarily in their phosphorylated form. These are termed nucleotides.

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The nucleotides found in DNA are The nucleotides found in DNA are unique from those of RNA in that the unique from those of RNA in that the ribose exists in the 2'-deoxy form ribose exists in the 2'-deoxy form and the abbreviations of the and the abbreviations of the nucleotides contain a d designation. nucleotides contain a d designation.

The monophosphorylated form of The monophosphorylated form of adenosine found in DNA adenosine found in DNA (deoxyadenosine-5'-monophosphate) (deoxyadenosine-5'-monophosphate) is written as is written as dAMPdAMP. .

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The nucleotide uridine is never found in The nucleotide uridine is never found in DNADNA and and thymine thymine

is almost exclusively found in DNA. is almost exclusively found in DNA. Thymine is found in tRNAs but not rRNAs nor Thymine is found in tRNAs but not rRNAs nor

mRNAs. mRNAs. There are several less common bases found There are several less common bases found

in DNA and RNA. in DNA and RNA. The primary modified base in DNA is 5-The primary modified base in DNA is 5-

methylcytosine.methylcytosine. A variety of modified bases appear in the A variety of modified bases appear in the

tRNAs. tRNAs. Many modified nucleotides are encountered Many modified nucleotides are encountered

outside of the context of DNA and RNA that outside of the context of DNA and RNA that serve important biological functions.serve important biological functions.

PolynucleotidesPolynucleotides

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Polynucleotides (e.g. DNA and RNA) are Polynucleotides (e.g. DNA and RNA) are formed by the condensation of more formed by the condensation of more nucleotides. nucleotides.

The condensation most commonly The condensation most commonly occurs between the alcohol of a 5'-occurs between the alcohol of a 5'-phosphate of one nucleotide and the 3'-phosphate of one nucleotide and the 3'-hydroxyl group of a second (adjacent hydroxyl group of a second (adjacent nucleotide), with the elimination of Hnucleotide), with the elimination of H22O, O, forming a forming a phosphodiester bond.phosphodiester bond.

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The formation of phosphodiester bonds in The formation of phosphodiester bonds in DNA and RNA exhibits directionality. DNA and RNA exhibits directionality.

The primary structure of DNA and RNA The primary structure of DNA and RNA (the linear arrangement of the (the linear arrangement of the nucleotides) proceedsnucleotides) proceedsin the 5' ----> 3' direction. in the 5' ----> 3' direction.

The common representation of the The common representation of the primary structure of DNA or RNA primary structure of DNA or RNA molecules is to write the nucleotide molecules is to write the nucleotide sequences from left to right synonymous sequences from left to right synonymous with the 5' -----> 3' direction as shown: with the 5' -----> 3' direction as shown:

5'- pG pA pT pC - 3'5'- pG pA pT pC - 3'

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Utilizing X-ray diffraction data, obtained Utilizing X-ray diffraction data, obtained from crystals of DNA, James Watson and from crystals of DNA, James Watson and Francis Crick proposed a model for the Francis Crick proposed a model for the structure of DNA.structure of DNA.

This model (subsequently verified by This model (subsequently verified by additional data) predicted that DNA additional data) predicted that DNA would exist as a helix of two would exist as a helix of two complementary anti parallel strands, complementary anti parallel strands, wound around each other in a rightward wound around each other in a rightward direction and stabilized by H-bonding direction and stabilized by H-bonding between bases in adjacent strands.between bases in adjacent strands.

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In the In the Watson-Crick modelWatson-Crick model, the bases are , the bases are in the interior of the helix aligned at a in the interior of the helix aligned at a nearly 90 degree angle relative to the axis nearly 90 degree angle relative to the axis of the helix. of the helix.

Purine bases form hydrogen bonds with Purine bases form hydrogen bonds with pyrimidines, in the crucial phenomenon of pyrimidines, in the crucial phenomenon of base pairing.base pairing.

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Experimental determination of molecule of Experimental determination of molecule of DNA has shown the concentration of adenine DNA has shown the concentration of adenine (A) is equal to thymine (T) and the (A) is equal to thymine (T) and the concentration of cytidine (C) is equal to concentration of cytidine (C) is equal to guanine (G). guanine (G).

This means that A will only base-pair with T, This means that A will only base-pair with T, and C with G. and C with G.

According to this pattern, known as According to this pattern, known as Watson-Watson-Crick base-pairingCrick base-pairing, the base-pairs composed , the base-pairs composed of G and C contain three H-bonds, whereas of G and C contain three H-bonds, whereas those of A and T contain two H-bonds. those of A and T contain two H-bonds.

This makes G-C base-pairs more stable than A-This makes G-C base-pairs more stable than A-T base-pairs. T base-pairs.

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(a) Base pairing A-T and G-C in the Watson-Crick model of DNA

(b) Distance between the base pair. The base shows the 0.34 nm distance between them.

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The antiparallelThe antiparallel nature of the helix stems nature of the helix stems from the orientation of the individual from the orientation of the individual strands. strands.

From any fixed position in the helix, one From any fixed position in the helix, one strand is oriented in the 5' ---> 3' direction strand is oriented in the 5' ---> 3' direction and the other in the 3' ---> 5' direction.and the other in the 3' ---> 5' direction.5’ 5’ C-G-A-T-A-G C-G-A-T-A-G 3’3’

3’ 3’ G-C-T-A-T-C G-C-T-A-T-C 5’5’

On its exterior surface, the double helix of On its exterior surface, the double helix of DNA contains two deep grooves between the DNA contains two deep grooves between the ribose-phosphate chains. ribose-phosphate chains.

These two grooves are of unequal size and These two grooves are of unequal size and termed the termed the majormajor and and minor groovesminor grooves. .

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The difference in their size is due to the The difference in their size is due to the asymmetry of the deoxyribose rings and the asymmetry of the deoxyribose rings and the structurally distinct nature of the upper surface structurally distinct nature of the upper surface of a base-pair relative to the bottom surface.of a base-pair relative to the bottom surface.

The double helix of DNA has been shown to The double helix of DNA has been shown to exist in several different forms, depending exist in several different forms, depending upon sequence content and ionic conditions of upon sequence content and ionic conditions of crystal preparation. crystal preparation.

IMPORTANT BIOLOGICAL IMPORTANT BIOLOGICAL FUNCTION OF NUCLEOTIDESFUNCTION OF NUCLEOTIDES

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Nucleotides have a number of Nucleotides have a number of important functions:important functions: precursor units for the synthesis of the precursor units for the synthesis of the

nucleic acids DNA and RNAnucleic acids DNA and RNA linkage of energy yielding reactions to linkage of energy yielding reactions to

those which require energythose which require energy activated intermediates in many activated intermediates in many

biosynthetic processesbiosynthetic processes synthesis of important coenzymes synthesis of important coenzymes

metabolic regulationmetabolic regulation

MEDICAL APPLICATIONS OF MEDICAL APPLICATIONS OF NUCLEOBASES, NUCLEOSIDES AND NUCLEOBASES, NUCLEOSIDES AND

NUCLEOTIDESNUCLEOTIDES

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Synthetic analogues of naturally Synthetic analogues of naturally occurring nucleobases, nucleosides or occurring nucleobases, nucleosides or nucleotides that can act as enzyme nucleotides that can act as enzyme inhibitors or replace naturally occurring inhibitors or replace naturally occurring nucleotides in nucleic acids are nucleotides in nucleic acids are increasingly being used in the increasingly being used in the chemotherapy of cancer and certain chemotherapy of cancer and certain other clinical conditions.other clinical conditions.

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59 CANCER CHEMOTHERAPYCANCER CHEMOTHERAPY

Example of synthetic analogues used in Example of synthetic analogues used in cancer chemotheraphy include cancer chemotheraphy include 5’-5’-fluorouracil, 6-mercaptopurine, and fluorouracil, 6-mercaptopurine, and cytosine arabinoside.cytosine arabinoside.

ANTIFUNGAL AGENTSANTIFUNGAL AGENTSFlucytosine Flucytosine (5-fluorocytosine) acts as an (5-fluorocytosine) acts as an antifungal agent through convertion to 5-antifungal agent through convertion to 5-fluoroacil in the fungal cells.fluoroacil in the fungal cells.

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60 ANTIVIRAL AGENTSANTIVIRAL AGENTS

Idoxuridine Idoxuridine (iododeoxyuridine) is effectively (iododeoxyuridine) is effectively used in the treatment of corneal infections by used in the treatment of corneal infections by herpes virus. It acts as a competitive herpes virus. It acts as a competitive inhibitors (via phosphorylated derivatives) of inhibitors (via phosphorylated derivatives) of the incorporation of thymidylic acid into DNA.the incorporation of thymidylic acid into DNA.

TREATMENT OF HYPERURICAEMIA AND GOUTTREATMENT OF HYPERURICAEMIA AND GOUTAllopurinol (4-hydroxypyrazolopyrimidine)Allopurinol (4-hydroxypyrazolopyrimidine), a , a structural analogue of hypoxanthine is widely structural analogue of hypoxanthine is widely used as an inhibotor of used as an inhibotor of de novode novo purine purine biosynthesis, and of biosynthesis, and of xanthinexanthine oxidase.oxidase.

References:References:

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Ahmad, Mushtaq – Ahmad, Mushtaq – Essentials of Medical BiochemistryEssentials of Medical Biochemistry, , Vol. 1, 6Vol. 1, 6thth ed. Merit Publishers, Multan, 1999. ed. Merit Publishers, Multan, 1999.

Lehninger, A.L. – Lehninger, A.L. – Biochemistry: The molecular basis of Biochemistry: The molecular basis of cell structure and function, cell structure and function, 33rdrd

ed. Worth Publishers ed. Worth Publishers Inc., N.Y., 1993.Inc., N.Y., 1993.

Murray, R.K., Granner. D.K., Mayes, P.A., and Rodwell, Murray, R.K., Granner. D.K., Mayes, P.A., and Rodwell, V.W. (eds) – V.W. (eds) – Harper’s Biochemistry, Harper’s Biochemistry, 2424thth ED. Appleton ED. Appleton and Lange, California, 1996.and Lange, California, 1996.

Stryer, L. – Stryer, L. – BiochemistryBiochemistry, 4, 4thth ed. W.H. Freeman and ed. W.H. Freeman and Co., N.Y., 1995.Co., N.Y., 1995.