NUCLEOTIDE STRUCTURE,

FUNCTIONS, METABOLISM
AND DNA REPLICATION
by
Assoc Prof Dr Gnanajothy Ponnudurai
LECTURE OUTCOME

• After completing this lecture you should be able to:

• To outline the basic structures of nucleosides, nucleotides and

nucleic acids and their functions.
• To describe briefly the denovo synthesis, salvage pathways and
degradation of nucleotides.
• To describe the disorders of nucleotide metabolism.
• To explain the role of folic acid in nucleotide biosynthesis
• To describe briefly the process of DNA replication with special
reference to telomerase function and DNA repair mechanisms.
BASIC STRUCTURE OF NUCLEOSIDE AND
NUCLEOTIDES
Components of nucleotides

• A nitrogenous base : purine or pyrimidine

• A pentose sugar : ribose or deoxyribose
• One or more phosphate groups

Nitrogenous base + pentose sugar = Nucleoside

Nitrogenous base + pentose sugar + phosphate = Nucleotide

Nucleotide

Phosphate Base

Pentose
sugar
Nitrogenous Base

i) Pyrimidines
Cytosine
Thymine

Uracil (U)

ii) Purines
Adenine
Guanine
Pentose Sugar

i) Ribose

ii) Deoxyribose

In ribose, X= OH ; In deoxyribose, X = H
 FUNCTIONS OF
NUCLEOTIDES
• Metabolic functions of nucleotides
• Building blocks of DNA and RNA .
• Medium of energy exchange inside cell, eg ATP.
• Nucleotides form components of coenzymes, eg. NAD+, FAD, CoA
• Metabolic regulators, eg. cAMP
• Activated intermediates, eg. UTP  UDP-glucose
• Allosteric modifiers, eg ATP, ADP, AMP
 The pentose required by nucleotide are
synthesized by the pentose phosphate
DENOVO pathway.

BIOSYNTH Source of
• The atoms in the purine ring
are contributed by
ESIS OF atoms in
glutamine, glycine, aspartate,
carbon dioxide , and formyl-
Purine ring
NUCLEOTI tetrahydrofolate (Formyl-
THF)

DES Source of • The atoms in the pyrimidine

atoms in ring are contributed by
Pyrimidine Aspartate, Glutamine, and
ring Carbon dioxide
 Source of Atoms in the Purine Ring

Glycine
CO2

Aspartate C N
N C
C Formyl-
C tetrahydrofolate
C
N N
Formyl-
tetrahydrofolate Glutamine
Source of Atoms in the Pyrimidine Ring

Glutamine Aspartate

HN

CO2 N
PURINE SYNTHESIS
Purine is synthesised
starting with ATP and Inosine
The purine base is ribose 5-phosphate
monophosphate
synthesised on the which forms
5’phosphorybosyl-1’- (IMP) is
ribose moiety.
pyrophosphate generated.
(PRPP).

AMP and GMP is Conversion of ribose

IMP is the phosphorylated moiety to deoxyribose
occur at diphosphate
precursor for to the level. dATP and dGTP
AMP and GMP. triphosphate is used for DNA
level. synthesis.
De novo
biosynthesis
of purine • Methotrexate, is an analog of
tetrahydrofolate (FH4)
• Inhibits reduction of
dihydrofolate to
tetrahydrofolate by inhibiting
dihydrofolate reductase.
• Methotrexate limits the
amount of tetrahydrofolate
available for purine synthesis,
thus slows down DNA
replication.
• Used as anti cancer drug, but
also toxic to all dividing cells.
PYRIMIDINE SYNTHESIS
The pyrimidine base is Aspartate is added to
Glutamine reacts with
synthesised prior to carbamoyl phosphate,
CO2 and ATP to from
addition of the ribose forming orotate (orotic
carbamoyl phosphate.
moiety. acid).

Orotate reacts with

PRPP forming UMP, UTP reacts with
which is glutamine to form CTP.
phosphorylated to UTP.
De novo synthesis
of pyrimidine • Thymidylate
synthase
catalyses
conversion of
dUMP to dTMP.
• 5-Fluorouracil
inhibits
thymidylate
synthase, thus
inhibiting
thymidine
formation
required for DNA
synthesis.
 SALVAGE PATHWAYS

• This pathway allows undegraded purine and pyrimidine from diet

or from the normal turnover of cellular nucleic acid, to be salvaged
and reutilized by body to form nucleotides. Results in energy
saving for cells.

• A critical step in the salvage pathway of purines is catalysed by the

enzyme Hypoxanthine Guanine Phosphoribosyltransferase
(HGPRT).
 CATABOLISM OF NUCLEOTIDES
• Degradation of nucleotides involves sequential removal of
phosphate groups and pentose sugar.
• Finally, excess purines and pyrimidines that cannot be utilized
through salvage pathways are degraded.

• Degradation of Purines
• Degradation of guanine produces xanthine.
• Degradation of adenine produces hypoxanthine, which is
oxidized to xanthine by xanthine oxidase.
• Xanthine is oxidized to uric acid by xanthine oxidase. Uric acid
is excreted in the urine.
Catabolism of purines

GMP IMP AMP

Guanosine
Adenosine
deaminase
Guanine Inosine Adenosine

Xanthine
oxidase
Xanthine Hypoxanthine
Adenosine deaminase deficiency causes
Xanthine
severe combined immunodeficiency (SCID)
oxidase
involving both T and B lymphocytes. The
adenosine that accumulates is toxic to both
Uric acid
T and B lymphocytes
 CATABOLISM OF NUCLEOTIDES

• Degradation of pyrimidines

• Uracil and cytosine are converted to -alanine, thymine is

converted to -aminoisobutyrate. CO2 and ammonia are also
produced.
• Products of pyrimidine degradation do not cause any problems
to the body.
DISORDERS OF PURINE
NUCLEOTIDE METABOLISM
Gout
• Caused by :
• increased conversion of purine bases to uric acid
• decreased excretion of uric acid by kidney

• Uric acid, which is very insoluble, accumulates, results in the precipitation of

sodium urate in the joints.

• Treatment is with allopurinol (chemically modified form of xanthine), which

binds to xanthine oxidase and decreases production of uric acid.
DISORDERS OF PURINE
NUCLEOTIDE METABOLSIM
• Lesch-Nyhan syndrome

• Caused by deficiency of the enzyme HGPRT

• Purine bases cannot be salvaged (i.e. cannot be reconverted to

nucleotides). Instead purines are degraded, forming excessive amount of
uric acid.

• Lesch-Nyhan syndrome is associated with gout, increased sodium urate,

mental retardation and self-mutilation.
DISORDERS OF PYRIMIDINE
NUCLEOTIDE METABOLSIM
Hereditary orotic aciduria

• In hereditary orotic aciduria, orotic acid is exreted in the urine because the
enzyme that converts it to UMP, orotate phosphorybosyl transferase and
orotidine 5’-phosphate decarboxylase, are defective.
• Pyrimidine cannot be synthesised and therefore normal growth does not
occur.
• Oral administration of uridine bypasses the metabolic block and provides the
body with a source of purimidine.
 Summary
• Structure of a nucleotide –Nitrogenous base (Pu/Py) + pentose sugar
(ribose/deoxyribose) + phosphate groups.
• Compounds that contribute purine ring are amino acids (aspartic acid, glycine,
glutamine), CO2 , fTHF.
Pyrimidine ring - aspartic acid, glutamine, CO2.
• Function of purine salvage pathway is to convert purine that result from cell turnover or
from diet into nucleotide.
• In denovo biosynthesis of purine nucleotide, purine base is synthesised on ribose.
In denove biosynthesis of pyrimidine nucleotide, pyrimidine
base is synthesised prior to addition of ribose.
• End product of purine catabolism is uric acid.
• Disorders of purine metabolism – Gout, Lesch Lyhan syndrome.
Disorder of pyrimidine metabolism – orotic aciduria
 Questions
• What differentiates a nucleoside from a nucleotide?
Addition of phosphate group(s) to nitrogenous base and pentose sugar

• A 50-year-old female patient complains of pain and swelling in her joints and
her laboratory results show hyperuricaemia. A disorder in the metabolism of
which of the following substances would MOST likely result in her condition?
A. Pyrimidines
B. Glucose
C. Purines
D. Amino acids
 Case scenario
A 4-year-old boy is brought to the paediatric clinic because of developmental delay. His
mother has mentioned that the boy has the tendency of biting his lips excessively and
hitting himself. Physical examination reveals scarring on his lips and swelling on the toes
and fingers. Laboratory tests reveal hyperuricemia.
• What disease is this boy suffering from?
Lesch-Nyhan Syndrome
• This disorder is due to deficiency of an enzyme. Name the enzyme.
HGPRT
• What pathway is this enzyme involved in?
Purine salvage pathway
• What is the inheritance pattern of this disease?
X-linked recessive
 NUCLEIC ACIDS

• There are two types of nucleic acids:

• i.         deoxyribonucleic acid (DNA)
• ii.        ribonucleic acid (RNA).

• Nucleic acids are linear polymers of monomers called

nucleotides.
NUCLEIC ACIDS

Type of Pentose Base Phosphate

nucleic acid sugar

DNA Deoxyribose Adenine (A) Phosphate

Guanine (G)
Cytosine (C)
Thymine (T)

RNA Ribose Adenine (A) Phosphate

Guanine (G)
Cytosine (C)
Uracil (U)
 Question from Lippincott Illustrated Reviews

The extent of DNA synthesis in a cell could most specifically be

determined by measuring the incorporation of radiolabelled
A. leucine.
B. phosphate.
C. ribose
D. thymidine
E. uracil
STRUCTURE OF A DNA
STRAND
• In DNA, nucleotides are joined by covalent
bonds called phosphodiester linkages
between phosphate on one nucleotide
and sugar of the next nucleotide.

• The bonding results in a 'sugar-phosphate-

sugar-phosphate' backbone.

• All along this sugar-phosphate backbone

are appendages consisting of the
nitrogenous bases.
 Hyd
DOUBLE HELIX

rog
en b
STRUCTURE OF

ond
DNA

s
• The DNA molecules of cells consists of two chains of nucleotides that form a double
helix.

• The two sugar-phosphate backbone are on the outside of the helix, and the
nitrogenous bases are paired in the interior of the helix.

• The two strands (chains) of nucleotides, are held together by hydrogen bonds between
the paired bases.
 DOUBLE HELIX STRUCTURE OF DNA

• The nitrogenous bases of the double helix are paired in specific combinations:

adenine (A) with thymine (T),

guanine (G) with cytosine (C).

• The two strands of the double helix are complementary. It is this feature of DNA
that makes possible the precise copying of genes that is responsible for inheritance.

• The strands are antiparallel

One strand runs in 5’to 3’ direction, the other strand runs in 3’ to 5’ direction.
DNA double helix Two complementary DNA sequences

DNA double helix

Source: Lippincott’s Illustrated reviews

Biochemistry 2011 (5th Ed)
 Question from Lippincott Illustrated Reviews

While studying the structure of a small gene that was recently sequenced during
the Human Genome Project, an investigator notices that one strand of the DNA
molecule contains 20 As, 25 Gs, 30 Cs, and 22Ts. How many of each base is found
in the complete double-stranded molecule?
A. A = 40, G = 50, C = 60, T = 44.
B. A = 44, G = 60, C = 50, T = 40.
C. A = 45, G = 45, C = 52, T = 52.
D. A = 50, G = 47, C = 50, T = 47.
E. A = 42, G = 55, C = 55, T = 42.
 DNA REPLICATION

What is DNA REPLICATION?

• DNA replication is semi-conservative.

In each molecule generated by replication:

- one strand is retained (conserved)
- one strand is newly synthesised
 Semi-conservative replication of DNA

Source: Lippincott’s Illustrated reviews

Biochemistry 2011 (5th Ed)
 DNA REPLICATION

Proteins Involved in DNA Replication

Topoisomerase

Helicase

Single-strand binding protein

 DNA REPLICATION

Other Proteins Involved in DNA Replication

Primase
DNA polymerase III Ligase
 DNA REPLICATION
Origins of replications

• Origins of Replication – special site where replication

begins.
• Replication is bidirectional - proceeds in both directions.
 Replication fork
Question

What is a replication fork?

Replication fork is where
the two strands of DNA
are unwinding and
separating, creating a “V”
which is where active
synthesis is taking place.

During replication nucleotides are added to the 3’ end of the strand

 TELOMERE AND TELOMERASE

• Telomeres are repetitive nucleotide sequences (TTAGGG) at the ends of eukaryotic

chromosomes
• Forms a cap - protects ends, maintains length
• Telomere is maintained by enzyme telomerase.
• Telomerase is active only in germ cells and stem cells.
• Direct relationship btw telomerase and aging, telomerase has the ability to prolong
life and cell division.
• Most somatic cells do not have sufficiently high levels of telomerase to maintain
the length of telomeres. Telomeres gradually shortens as cells age leading to cell
death.
• Cancer cells express abnormally high levels of telomerase, allowing them to
continue dividing
 Question from Lippincott Illustrated Reviews

Telomeres are complexes of DNA and protein that protect the ends of linear
chromosomes. In most normal human somatic cells, telomeres shorten with
each division. In stem cells and in cancer cells, however, telomeric length is
maintained. In the synthesis of telomeres:
A. telomerase, a ribonucleoprotein, provides both the RNA and the polymerase
needed for synthesis.
B. the RNA of telomerase serves as a primer.
C. the polymerase of telomerase is a DNA-directed DNA polymerase.
D. the shorter, 3'→5' strand gets extended.
E. the direction of synthesis is 3'→5'.
 DNA DAMAGE AND REPAIR

• DNA damage can be due to

• Exogenous source
• Radiation (eg UV),

• Chemical substance
• alkylating substances – methylate or ethylate bases, causing them to pair with other bases than the usual
• Intercalating substances –embed between the DNA base pairs , causing replication to stopand increase
risk of strand breaks (eg ethidium bromide)

• Endogenous source
• replication errors
• chemical instability (eg spontaneous deamination of cytosine to uracil)

• Repair enzymes ( nuclease, DNA polymerase, DNA ligase) fix DNA damage
 DNA DAMAGE AND REPAIR

Single stranded DNA repair

Repair enzymes ( nuclease, DNA polymerase, DNA ligase) fix DNA damage

Base excision repair Nucleotide excision

Mismatch repair
repair
• Damage can be • Damage caused by • UV light can cause two
caused by chemical chance/replication adjacent nucleotides
agents (e.g. tobacco), errors to stick together 
naturally-occurring • Usually occurs during distorting DNA
compounds (i.e. H2O2) replication double-helix structure
or environmental • Occur after
exposures. replication.
• Occur after
replication.
Nucleotide excision repair of thymine dimers

UV light causes
formation of thymine
dimers.

Xeroderma
pigmentosum is a skin
disorder, caused by an
inherited defect in the
repair enzyme
involved in removal of
thymine dimers. Source: Lippincott’s Illustrated
reviews Biochemistry 2011 (5th Ed)

Leads to skin cancer.

 Summary
• DNA
• is a hereditary molecule
• consists of sugar-phosphate backbone and nitrogenous bases
• is double-helix and anti-parallel
• Has complementary base-pairing: A with T, C with G
• DNA replication is semi-conservative.
• DNA is synthesized from the 5’ to 3’ end of the new strand.
• DNA replication consists of unwinding, synthesizing (incl. primer-replacement) and ligation.
• Multiple origins of replications.
• Leading strand replicates forming a continuous strand.
• Lagging strand needs multiple Okazaki fragments to be synthesized before they are being
ligated to produce the new strand.
 Question from Lippincott Illustrated Reviews

A 10-year-old girl is brought to the dermatologist by her parents. She has

many freckles on her face, neck, arms, and hands, and the parents report
that she is unusually sensitive to sunlight. Two basal cell carcinomas are
identified on her face. Which of the following processes is most likely to be
defective in this patient?

A. Repair of double-strand breaks.

B. Removal of mismatched bases from the 3'-end of Okazaki fragments.
C. Removal of pyrimidine dimers from DNA.
D. Removal of uracil from DNA.
 References
Amboss:

Nucleotides, DNA and RNA

https://next.amboss.com/us/article/oo001S

Purine and pyrimidines

https://next.amboss.com/us/article/6o0j1S

DNA replication and repair

https://next.amboss.com/us/article/Ko0U1S#Zf242ab2ee618bfce0bc6dc3d31a33740

Further reading
• Champe, P.C., Harvey, R.A. (2011)Lippincott’s Illustrated Reviews- Biochemistry, 5 TH Edition. Chapter 29, page 395

• Telomers and telomerase https://www.khanacademy.org/science/biology/dna-as-the-genetic-material/dna-replication/

a/telomeres-telomerase
If you have any queries, please contact gnanajothy_ponnudurai@imu.edu.my