dna sythensis

1. Eukaryotic and Prokayrotic
DNA Replication
Dr. S.B. MADA
Department of Biochemistry
KIU-Western Campus

2. Metabolism is a term referring to both anabolism and catabolism
Catabolism means breakdown of large biomolecule into smaller ones.
Anabolism refers to the formation of large biomolecule from the
precursor molecules.
The metabolic requirements for the nucleotides and their cognate bases
can be met by both dietary intake and synthesis de novo from low
molecular weight precursors.
The ability to salvage nucleotides from sources within the body
alleviates any nutritional requirement for nucleotides.
Therefore, the purine and pyrimidine bases are not required in the diet.
The salvage pathways are a major source of nucleotides for synthesis of
DNA, RNA and enzyme co-factors.
Metabolism

3. Nitrogenous Bases
• Planar, aromatic, and heterocyclic
• Two types: Purine or Pyrimidine
• Numbering of bases is “unprimed and
anticlockwise”

4. Nucleic Acid Bases
Purines Pyrimidines

5. Sugars
• Pentoses (5-C sugars)
• Numbering of sugars is “primed and clockwise”

6. Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group

7. Nucleosides
• Result from linking one of the sugars with
a purine or pyrimidine base through an N-
glycosidic linkage
– Purines bond to the C1’ carbon of the sugar at
their N9 atoms
– Pyrimidines bond to the C1’ carbon of the
sugar at their N1 atoms

8. Phosphate Groups
• Mono-, di- or triphosphates
• Phosphates can be bonded to either C3 or
C5 atoms of the sugar

9. Nucleotides
• Result from linking one or more phosphates
with a nucleoside onto the 5’ end of the
molecule through esterification

10. Nucleosides

11. Nucleotides
• RNA (ribonucleic acid) is a polymer of
ribonucleotides
• DNA (deoxyribonucleic acid) is a polymer
of deoxyribonucleotides
• Both deoxy- and ribonucleotides contain
Adenine, Guanine and Cytosine
– Ribonucleotides contain Uracil
– Deoxyribonucleotides contain Thymine

12. Biological function of Nucleotides
i. Nucelotides are monomers or building blocks of
ribonucleic acid (RNA) and deoxyribonucleic acid
(DNA)
ii. Nucleotides serve as important energy carriers e.g.
ATP is the universal currency of energy in biological
systems
iii. Nucleotides are important components of coenzymes
e.g. FAD, NAD+ and Coenzyme A
iv. Nucleotides serve as activated intermediates in many
biosynthesis e.g. UDP-glucose in glycogen synthesis.
v. Nucleotides serve as secondary messengers (cAMP
and cGMP) in biological systems

15. Genes in DNA contain information
needed to synthesize mRNA in the
nucleus
The mRNA produced is translated
into proteins in the cytosol.
The protein synthesized is then
transported to various tissues and
organs for cellular function .
Overview

16. Gene expression: DNA  RNA  Protein
DNA
DNA
RNA
Protein
Replication
Transcription
Translation
Degradation
Degradation
Initiation
Elongation
Termination
Processing
Export
Initiation
Elongation
Termination
Processing
Targeting
Overview

17. DNA Replication
Process of duplication of the entire genome prior to cell
division
Biological significance
• extreme accuracy of DNA replication is necessary in
order to preserve the integrity of the genome in
successive generations
• Replication rate in eukaryotes is slower resulting in a
higher fidelity/accuracy of replication in eukaryotes
than in prokaryotes.

18. Four requirements for DNA to be
genetic material
Must carry information
– Cracking the genetic code
Must replicate
– DNA replication
Must allow for information to change
– Mutation
Must govern the expression of the phenotype
– Gene function

19. When & why does DNA have to replicate?
 Replicates so that there will be enough genetic information for
the cell to divide
 DNA replication occurs during the S phase of the cell cycle.
 DNA Replicates inside the nucleus In each cell division, cell must
copy its entire DNA So each daughter cell gets a complete copy
 Rate of synthesis
– Bacteria = 1000 bases per second
– Mammals = 100 bases per second
 Problem - with a single replication origin in DNA
– Bacteria genome is 4 x 106. Takes 20 minutes to copy.
– Human is 3.2 x 109. Would take 10,000 times longer.

20. Genetic Information Must Be Accurately
Copied Every Time a Cell Divides
• Replication has to be extremely accurate:
• 1 error/million bp leads to 6400 mistakes
every time a cell divides, which would be
catastrophic.
• Replication also takes place at high speed:
• E. coli replicates its DNA at a rate of 1000
nucleotides/second.

21. • Conservative replication model
• Dispersive replication model
• Semiconservative replication model
Proposed DNA Replication Models

22. Proposed DNA Replication Models

23. • Two isotopes of nitrogen:
• 14N common form; 15N rare heavy form
• E. coli were grown in a 15N media first, then
transferred to 14N media.
• Cultured E. coli were subjected to
equilibrium density gradient centrifugation.
Meselson and Stahl’s Experiment

25. Enzymes involves in DNA
Replication
DNA Polymerase - Joins adjacent
nucleotides to each other.
Primase - Provides an RNA primer to
start polymerization
Ligase - Joins adjacent DNA strands
together (fixes “nicks”)

26. Helicase - Unwinds the DNA and melts it
Single Strand Binding Proteins - Keep the
DNA in single stranded form after it has
been melted by helicase
Gyrase - A topisomerase that Relieves
torsional strain in the DNA molecule
Telomerase - Finishes off the ends of
DNA strands
Enzymes involves in DNA
Replication cont…

27. Eukaryotic DNA Polymerases
Enzyme Location Function
• Pol  (alpha) Nucleus DNA replication
– includes RNA primase activity, starts DNA strand
• Pol  (gamma) Nucleus DNA replication
– replaces Pol  to extend DNA strand, proofreads
• Pol  (epsilon) Nucleus DNA replication
– similar to Pol , shown to be required by yeast mutants
• Pol  (beta) Nucleus DNA repair
• Pol  (zeta) Nucleus DNA repair
• Pol  (gamma) Mitochondria DNA replication

28. First discovered in 1956 by Kornberg
In E.coli.
Bacteria have 3 types
 DNA Pol I, II, and III
DNA Pol III involved in replication of DNA
DNA Pol I involved in repair
Prokaryotic DNA Polymerases

29. 3
Polymerase III
5’ 
3’
Leading strand
base pairs
5’
5’
3’
3’
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
Helicase
+
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2
Polymerase III
Lagging strand
Okazaki Fragments
1
RNA primer replaced by polymerase I
& gap is sealed by ligase

30. Bacterial DNA polymerases cont…
1955: Arthur Kornberg
Worked with E. coli.
Discovered the mechanisms of DNA synthesis.
Four components are required:
1. dNTPs: dATP, dTTP, dGTP, dCTP
(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase I (formerly the Kornberg enzyme)
(DNA polymerase II & III discovered soon after)
4. Mg 2+ (optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)
Arthur Kornberg, a Nobel prize

31. • Circular E. coli; DNA has single origin of
replication forming a replication fork, usually a
bidirectional replication
• Virus has single origin of replication
• Eukaryotic cells; have multiple of origin of
replication ; a typical replicon: 200,000 ~
300,000 bp in length
Origin of Replication

32. Stages of DNA Replication
– Initiation
• Enzymes and proteins bind to DNA and open up
double helix
• Prepare DNA for complementary base pairing
– Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
– Termination
• Proteins release the replication complex

33. • Requirements of replication:
• A template strand
• Raw material: nucleotides (dNTPs)
• Enzymes and other proteins
• Mg 2+
• Direction of replication:
• DNA polymerase add nucleotides only to the 3′ end of a
growing strand.
• Thus, replication occur only at 5′3′ direction.
Eukaryotic DNA Replication

34. DNA Replication

35. • Direction of replication:
• Leading strand: undergoes continuous
replication
• Lagging strand: undergoes discontinuous
replication
• Okazaki fragment: these are short DNA
fragments synthesized in discontinuous
manner forming the lagging strand and
joined together by DNA ligase
Eukaryotic DNA Replication

36. Prokaryotic /Bacterial DNA
Replication
• Initiation: 245 bp in the oriC (single origin
replicon); an initiation protein
• Helicase unwinding of DNA is performed by
• Gyrase removes supercoiling ahead of the replication
fork. Single stranded DNA is prevented from annealing by
single stranded binding proteins.
• Primers: an existing group of RNA nucleotides with a 3′-
OH group to which a new nucleotide can be added;
usually 10 ~ 12 nucleotides long
Primase: RNA polymerase

37. Bacterial DNA Replication
• Elongation: carried out by DNA polymerase
III
• Removing RNA primer: DNA polymerase I
• DNA ligase: connecting nicks after RNA
primers are removed
• Termination: when a replication fork meets
termination signal/protein

38. Bacterial DNA Replication

39. Mistakes during Replication
• Base pairing rules must be maintained
– Mistake can leads to genome mutation, may have
consequence on daughter cells
• Only correct pairings fit into the polymerase active site
• If wrong nucleotide is included will not fit and DNA
Polymerase will pause for repair to occur
Proofreading:
 DNA polymerase I: Posess 3′5′ exonuclease activity
removes the incorrectly paired nucleotide.
 DNA POL III Adds correct nucleotide and proceeds down
the chain again in the 5’  3’ direction
 Mismatch repair: correcting errors after replication is
complete

40. Proofreading
High Fidelity DNA Replication
Error rate= 1 mistake/109 nucleotides afforded by complementary base
pairing and proof-reading capability of DNA polymerase

41. THANK YOU