DNA replication occurs through a semiconservative process where the parental DNA strands separate and act as templates for the synthesis of new complementary strands. Key experiments by Meselson and Stahl provided evidence for this semiconservative model. DNA polymerase, discovered by Arthur Kornberg in 1955, is the main enzyme that catalyzes DNA synthesis. It requires DNA templates, dNTPs, and magnesium ions to carry out the step-wise addition of nucleotides in the 5' to 3' direction to form new DNA strands.

1. DNA Replication
Harshraj Subhash Shinde

2. Watson and Crick’s model of replication
• The base-pairing principal inherent for Watson-Crick
model suggests that the two new strands are copied
from two old strands.

3. Models of DNA replication
• Parent molecule remains the same
• Completely new copy of the double helix is made
• Parent strands separate and serve as
templates for new strand synthesis
• Hybrid molecules are made
• New strands contain a mixture of old
molecules and newly synthesized
molecules

4. 1958: Matthew Meselson & Frank Stahl’s Experiment
Equilibrium density gradient centrifugation

5. 1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication

6. 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 (Kornberg enzyme)
4. Mg 2+ (optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University)

7. DNA polymerase
• DNA polymerases are important enzymes involved in DNA
• replication.
• 􀀹Three polymerases have been purified from E.coli.
• 􀀹In addition to important role in filling the gaps between
• Okazaki fragments, DNA polymerase I is the most
important enzyme for gap filling during DNA repair.
• 􀀹DNA polymerase II functions in gap filling and appears to
facilitate DNA synthesis directed by damaged templates.
• 􀀹DNA polymerase III catalyzes chain elongation at the
• growing fork of E. coli.

8. Three main features of the DNA synthesis reaction:
1. DNA polymerase I catalyzes formation of phosphodiester bond
between 3’-OH of the deoxyribose (on the last nucleotide) and
the 5’-phosphate of the dNTP.
• Energy for this reaction is derived from the release of two of
the three phosphates of the dNTP.
2. DNA polymerase “finds” the correct complementary dNTP at
each step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate
3. Direction of synthesis is 5’ to 3’
DNA polymerase
Image credit:
Protein Data Bank

9. DNA elongation

10. There are many different types of DNA polymerase
Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) #Copies
I Yes Yes Yes 400
II Yes Yes No ?
III Yes Yes No 10-20
•3’ to 5’ exonuclease activity = ability to remove nucleotides from the
3’ end of the chain
•Important proofreading ability
•Without proofreading error rate (mutation rate) is 1 x 10-6
•With proofreading error rate is 1 x 10-9 (1000-fold decrease)
•5’ to 3’ exonuclease activity functions in DNA replication & repair.

15. Number of replication origins
Prokaryotes
• 1 replication origin per chromosome
• replication rate = 500 nucleotides per sec.
Eukaryotes
• multiple replication sites on each
chromosome.
• replication rate = 50 nucleotides per sec.

16. DNA Replication Origin of E.coli
• E. coli replication origin oriC is an ≈240bp DNA segment
present at the start
• site for the replication of the E. coli chromosomal DNA.
• Plasmids or any other circular DNAs containing oriC are
capable of independent and controlled replication in E.
coli cells.
• DnaA protein initiates replication.
• In addition, the E. coli genome contains a segment of
DNA with a relatively high A+T content adjacent to the
oriC.

19. MCQs

20. • DNA polymerase is discovered by
A. Watson and Crick
B. Crickmore
C. Meselson & Stahl’s
D. Korenberg
•

21. • Korenberg

22. • DNA polymerase is discovered in
a. 1954
b. 1955
c. 1958
d. 1962

23. • 1955

24. 1.Semi conservative replication of DNA was
first demonstrated in
a.Drosophila melanogaster
b.E. Coli
c.Arabidopsis thaliana
d.Human

25. • E. Coli

26. 1.Mode of DNA replication in E. Coli is
a.Conservative and unidirectional
b.Semi conservative and unidirectional
c.Conservative and bidirectional
d.Semi conservative and bidirectional

27. • Semi conservative and bidirectional

28. 1.When DNA replication starts
a.The phosphodiester bond between adjacent
nucleotides breaks
b.The bond between nitrogen base and
deoxyribose breaks
c. The hydrogen bond between the nucleotides
of two strand breaks
d.The leading strand produces okazaki fragment

29. • The hydrogen bond between the nucleotides
of two strand breaks

30. • Which of the following enzyme require ATP or
NAD for reaction
a.DNA ligase
b.DNA polymerase
c.DNA gyrase
d.Endonucleases

31. • DNA ligase

32. 1.During the replication of DNA, the synthesis
of DNA on lagging strand takes place in
segment, these segments are called
a.Satellite segment
b.Okazaki segment
c.Kornberg segment
d.Double helix segment

33. • Okazaki segment

34. 1.True replication of DNA takes place due to
a.Complementary base pairing rule
b.Hydrogen bonding
c.Phosphodiester bonding
d.Transcription and translation

35. • Complementary base pairing rule

36. 1.Unwinding of DNA Is done by
a.Topoisomerase
b.DNA polymerase
c.Helicase
d.Ligase

37. • Helicase

38. 1.DNA replication is specifically measured by
estimating the incorporation of radio labelled
a.Adenine
b.Uracil
c.Cytosine
d.Thymine

39. • Thymine

40. 1.The elongation of leading strand during DNA
synthesis
a.Progresses away from replication fork
b.Occurs in 3’ – 5’ direction
c.Depend on the action of DNA polymerase
d.Produces okazaki fragments

41. • Depend on the action of DNA polymerase

42. 1.Which of the following is not needed for DNA
replication?
a. Ribosomes
b. DNA
c. Nucleotides
d. Enzymes

43. • Ribosomes

44. • Experimental proof for semiconservative
mode of replication is give by
A. Watson and Crick
B. Meselson & Stahl’s
C. Korenberg
D. Khurana

45. • Meselson & Stahl’s

46. • Idea of DNA replication firstly proposed by
A. Watson and Crick
B. Crickmore
C. Meselson & Stahl’s
D. Korenberg

47. • Watson and Crick

48. • Rate of DNA replication is
a. Faster in prokaryotes than Eukaryotes
b. Faster in Eukaryotes than prokaryotes
c. Same in both Eukaryotes & prokaryotes
d. None of these

49. • Faster in prokaryotes than Eukaryotes

50. • Which of the following is E. coli origin of
replication
a. oriA
b. oriB
c. oriC
d. oriD

51. • oriC

52. • Which of the following is origin of replication
of yeast
a. Ori C
b. ARS
c. ARU
d. Ori A

53. • ARS

54. • Which of the following protein initiates DNA
replication in E.coli
a. DnaA
b. DnaB
c. SSB
d. SSA

55. • DnaA

56. • DNA polymerase is also known as
a. Kornberg's Enzyme
b. RNA polymerase
c. dNTPs adding and removing enzyme
d. PCR enzyme

57. • Kornberg's Enzyme

58. 1.Which of the following enzyme is used for
joining of bits of DNA?
a.DNA ligase
b.DNA polymerase
c.DNA gyrase
d.Endonucleases

59. • DNA ligase

60. • Which of the following enzyme plays
important role in gap filling DNA repair
mechanism
a. DNA polymerase I
b. DNA polymerase II
c. DNA polymerase III
d. RNA polymerase I

61. • DNA polymerase I