NUCLEIC ACID

METABOLISM
Pyrimidines
Biosynthesis
The Bases: Pyrimidines
 PYRIMIDINE
BIOSYNTHESIS
 1.PRPP(Phosphoribosylm Pyrophostate)
Synthetase

ATP+ Ribose 5-Phosphate PRPP

 2. Carbamoyl Phosphate Synthetase
Glutamine+ CO2 CPS II
+ 2 ATP Carbamoyl Phosphate + 2 ADP + P
3. Aspartate Carbamoyl Transferase

Carbomoyl phosphate + Aspartate ATcase

Carbomoyl Aspartate+ P
4.Dihdroorotate Dehydrogenase: the only
motochondrial enzymes in the pathway
Dihydroorotate+NAD DHOA
Orolate+NADH+ H
5. Orotate Phosphoribosyltransferase
Orotate
Orotate + PRPP phosphoribosyl OMP+ PP
transferase
 6. OMP Decarboxylase
OMP
OMP Decarboxylase UMP+CO2
(orotidine monophosphate) (uridine monophosphate)
 URIDINE CYTIDINE
UMP KINASE UDP
(Uridine Monophosphate) (Uridine Diphosphate)
 NUCLEOSIDE DIPHOSPHATE
UDP KINASE UTP
(uridine Diphosphate) ( Uridine Triphosphate)
 URIDINE CYTIDINE
UTP KINASE CTP
(Uridine Triphosphate) (cytidine Triphosphate)
 Regulation of Pyrimidine
Synthesis
• The primary site of regulation is carbamoyl
phosphate synthetase II (glutamine) which
is allosterically inhibited by UTP. Elevated
PRRP increase the CPS- II activity to help
control PRPP levels. Feedback
inhibition( control) is provided by TDP
inhibition of PRPP synthesis and UMP
inhibition of OMP
[7

7. Deoxyribonucleotide Synthesis

Synthesis of Thymidine nucleotides first requir

es deoxyribonucleotide synthesis. The enzym
e responsible for this step is
Ribonucleotide Reductase.
 RIBONUCLEOTIDE
UDP REDUCTASE dUDP
(Uridine diphosphate) (deoxyuridine Diphosphate)
 dUDP UMP Kinase
dUMP
(deoxyuridinediphosphate) (deoxyuridine monophosphate)
 dUMP Thymidylate
synthase TMP
(deoxyuridine monophosphate) (thymidine monophosphate)
 TMP(dTMP) Dtmp kinase TDP (dTDP)

(thymidine monophosphate) (thymidine diphosphate)

 RIBONUCLEOTIDE:
(OMP, UMP, UDP, UTP, CTP)

DEOXYRIBONUCLEOTIDE:
(dUDP, dUMP, dTMP, dTDP)
8. A deficiency of Adenosine Deaminase
(ADA) decreases metabolism of
deoxyadenosine causing dATP to
accumulate
9. Similarly a deficiency of Purine
Nucleoside Phosphorylase causes
accumulation of dGTP. In both cases
Ribonucleotide Reductase is inhibited by
accumulation of these nucleotides.
10. Synthesis of thymidylate requires uridine
nucleotide to be converted to the deoxy
form.
 Pyrimidine Salvage and
Metabolism
• Pyrimidine Phosphoribosyl Transferase:
Pyrimidine base + PRPP
Nucleoside monophosphate + PP

• Nucleoside kinase
nucleoside+ ATP Nucleotide
+ADP
DNA SYNTHESIS
 DNA SYNTHESIS

The discovery of the double

helical nature by Watson
and Crick explained how
genetic information could
duplicated and passed on to
succeeding generation.
• Maurice Wilkins and Rosalind Franklin were
using a technique called X-ray crystallography to
study molecular structure
• Rosalind Franklin produced a picture of the DNA
molecule using this technique
 DNA Synthesis
• DNA replication occurs during S-phase and is
initiated at origins of replication. It is a semi-
conservative in nature
• Replication occurs at replication fork
• DNA is only synthesised in the 5’ to 3’ direction
• Replication required coordination between
helicase, primase, SSBPs, two polymerases,
clamps, loaders, topisomerases, ligases and
RNA primase
DNA Synthesis
 REQUIREMENT FOR DNA
SYNTHESIS
Four Basic Component

• Substrate
• Template
• Primer
• Enzymes
Substrate
Four deoxyribonucleotide triphoshate
(dNTP) are required for DNA synthesis.
These are
•dATP( deoxyadenosine Triphosphate)
•dGTP( deoxyguanosine Triphosphate)
•dTTP( deoxythymidine Triphosphate)
•dCTP(deoxycytidine Triphosphate)
(dATP) (dTTP)

(dGTP) (dCTP)
Template
• The nucleotide that is to be incorporated
into the growing DNA chain is selected by
base pairing with the template strand of
the DNA.
Primer
• The enzyme that synthesizes DNA, DNA
polymerase, can only add nucleotide to an
already existing strand or primer of DNA or
RNA that is based paired with the template
• short strand of DNA or RNA generally
about 18-22 bases. It is the starting point
of DNA synthesis
 Enzymes
• DNA polymerase is an enzyme required for the
covalent joining of the incoming nucleotide to the
primer.
I. Prokaryotes
a. DNA polymerase I- repair
b. DNA polymerase II- clean up Okazaki fragment
c. DNA polymerase III- main polymerase
II. Eukaryotes
a. DNA polymerase a- lagging strand priming
b. DNA polymerase b- repair
3 STAGES OF DNA
REPLICATION
Initiation:
(1)Helicase unwinds the double-stranded DNA
(2)SSBPs bind to single strand DNA to keep DNA from
reannealing
(3)Topoisomerase (DNA gyrase) relaxes the twisting tension.
(4)Primase synthesizes short segments of RNA to create a
primer
(5)RNA Primer provides the 3’OH needed by DNA polymerase III
(6)DNA polymerase III catalyzes the formation of
phosphodiester bonds when nucleotides are added
DNA Synthesis
Elongation:
(1) DNA polymerase III adds nucleotide triphosphates
complementary to the DNA template starting from the 3’OH end
of the primer
☞ Complementary strands elongate from 5’ to 3’ as nucleotides are paired with the
template
☞ Complementary strand synthesized using the 3’ to 5’ templates elongates in the
same direction as the unwinding hence it is called leading strand
☞ Complementary strand of the 5’ to 3’ template is synthesized also in the 5’ to 3’
direction but opposite the direction of the unwinding hence it is called lagging strand
(2) DNA polymerase I proofreads the complementary strand and
removes the primer starting from the 3’ to 5’ direction. It also
replaces the primers with DNA nucleotides.
DNA Synthesis
Termination:
(1)DNA ligase seals or joins the nicks (break between the sugar-
phosphate backbone)
Other factor required for DNA Synthesis
• Origin
origin are unique DNA sequence that are
recognized by a protein that builds the replisome
Trans- acting Factor
Origin- binding protein- binds and partially
denature the origin DNA.
Helicase- unwind double stranded DNA.
Topoisomerase- remove the positive supercoils that
form as the fork is unwinded by the helicase.
• Single- stranded DNA binding protein (SSB)
enhances the activity of the helicase and
prevent the unwound DNA from renaturing.
• Primase
synthesize the RNA primer required for
initiating leading and lagging strand synthesis.
• DNA polymerase III
read each parental strand also called template,
catalyzed the polymerization of a
complementary daughter strand
• RNaseH
remove RNA portion from the okazaki
fragment
• ligase
seals the nicks after filling in the gaps left by
DNA polymerase.