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DNA Replication
Harshraj Subhash Shinde
Watson and Crick’s model of replication
• The base-pairing principal inherent for Watson-Crick
model suggests that the two...
Models of DNA replication
• Parent molecule remains the same
• Completely new copy of the double helix is made
• Parent st...
1958: Matthew Meselson & Frank Stahl’s Experiment
Equilibrium density gradient centrifugation
1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication
1955: Arthur Kornberg
Worked with E. coli.
Discovered the mechanisms of DNA synthesis.
Four components are required:
1. dN...
DNA polymerase
• DNA polymerases are important enzymes involved in DNA
• replication.
• 􀀹Three polymerases have been purif...
Three main features of the DNA synthesis reaction:
1. DNA polymerase I catalyzes formation of phosphodiester bond
between ...
DNA elongation
There are many different types of DNA polymerase
Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’)...
Number of replication origins
Prokaryotes
• 1 replication origin per chromosome
• replication rate = 500 nucleotides per s...
DNA Replication Origin of E.coli
• E. coli replication origin oriC is an ≈240bp DNA segment
present at the start
• site fo...
MCQs
• DNA polymerase is discovered by
A. Watson and Crick
B. Crickmore
C. Meselson & Stahl’s
D. Korenberg
•
• Korenberg
• DNA polymerase is discovered in
a. 1954
b. 1955
c. 1958
d. 1962
• 1955
1.Semi conservative replication of DNA was
first demonstrated in
a.Drosophila melanogaster
b.E. Coli
c.Arabidopsis thalian...
• E. Coli
1.Mode of DNA replication in E. Coli is
a.Conservative and unidirectional
b.Semi conservative and unidirectional
c.Conserv...
• Semi conservative and bidirectional
1.When DNA replication starts
a.The phosphodiester bond between adjacent
nucleotides breaks
b.The bond between nitrogen ba...
• The hydrogen bond between the nucleotides
of two strand breaks
• Which of the following enzyme require ATP or
NAD for reaction
a.DNA ligase
b.DNA polymerase
c.DNA gyrase
d.Endonucleases
• DNA ligase
1.During the replication of DNA, the synthesis
of DNA on lagging strand takes place in
segment, these segments are called
...
• Okazaki segment
1.True replication of DNA takes place due to
a.Complementary base pairing rule
b.Hydrogen bonding
c.Phosphodiester bonding...
• Complementary base pairing rule
1.Unwinding of DNA Is done by
a.Topoisomerase
b.DNA polymerase
c.Helicase
d.Ligase
• Helicase
1.DNA replication is specifically measured by
estimating the incorporation of radio labelled
a.Adenine
b.Uracil
c.Cytosine...
• Thymine
1.The elongation of leading strand during DNA
synthesis
a.Progresses away from replication fork
b.Occurs in 3’ – 5’ direct...
• Depend on the action of DNA polymerase
1.Which of the following is not needed for DNA
replication?
a. Ribosomes
b. DNA
c. Nucleotides
d. Enzymes
• Ribosomes
• Experimental proof for semiconservative
mode of replication is give by
A. Watson and Crick
B. Meselson & Stahl’s
C. Kore...
• Meselson & Stahl’s
• Idea of DNA replication firstly proposed by
A. Watson and Crick
B. Crickmore
C. Meselson & Stahl’s
D. Korenberg
• Watson and Crick
• Rate of DNA replication is
a. Faster in prokaryotes than Eukaryotes
b. Faster in Eukaryotes than prokaryotes
c. Same in ...
• Faster in prokaryotes than Eukaryotes
• Which of the following is E. coli origin of
replication
a. oriA
b. oriB
c. oriC
d. oriD
• oriC
• Which of the following is origin of replication
of yeast
a. Ori C
b. ARS
c. ARU
d. Ori A
• ARS
• Which of the following protein initiates DNA
replication in E.coli
a. DnaA
b. DnaB
c. SSB
d. SSA
• DnaA
• DNA polymerase is also known as
a. Kornberg's Enzyme
b. RNA polymerase
c. dNTPs adding and removing enzyme
d. PCR enzyme
• Kornberg's Enzyme
1.Which of the following enzyme is used for
joining of bits of DNA?
a.DNA ligase
b.DNA polymerase
c.DNA gyrase
d.Endonucle...
• DNA ligase
• Which of the following enzyme plays
important role in gap filling DNA repair
mechanism
a. DNA polymerase I
b. DNA polyme...
• DNA polymerase I
Dna replication
Dna replication
Dna replication
Dna replication
Dna replication
Dna replication
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Dna replication

DNA replication

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Dna replication

  1. 1. DNA Replication Harshraj Subhash Shinde
  2. 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. 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. 4. 1958: Matthew Meselson & Frank Stahl’s Experiment Equilibrium density gradient centrifugation
  5. 5. 1958: Matthew Meselson & Frank Stahl’s Experiment Semiconservative model of DNA replication
  6. 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. 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. 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. 9. DNA elongation
  10. 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.
  11. 11. 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.
  12. 12. 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.
  13. 13. MCQs
  14. 14. • DNA polymerase is discovered by A. Watson and Crick B. Crickmore C. Meselson & Stahl’s D. Korenberg •
  15. 15. • Korenberg
  16. 16. • DNA polymerase is discovered in a. 1954 b. 1955 c. 1958 d. 1962
  17. 17. • 1955
  18. 18. 1.Semi conservative replication of DNA was first demonstrated in a.Drosophila melanogaster b.E. Coli c.Arabidopsis thaliana d.Human
  19. 19. • E. Coli
  20. 20. 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
  21. 21. • Semi conservative and bidirectional
  22. 22. 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
  23. 23. • The hydrogen bond between the nucleotides of two strand breaks
  24. 24. • Which of the following enzyme require ATP or NAD for reaction a.DNA ligase b.DNA polymerase c.DNA gyrase d.Endonucleases
  25. 25. • DNA ligase
  26. 26. 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
  27. 27. • Okazaki segment
  28. 28. 1.True replication of DNA takes place due to a.Complementary base pairing rule b.Hydrogen bonding c.Phosphodiester bonding d.Transcription and translation
  29. 29. • Complementary base pairing rule
  30. 30. 1.Unwinding of DNA Is done by a.Topoisomerase b.DNA polymerase c.Helicase d.Ligase
  31. 31. • Helicase
  32. 32. 1.DNA replication is specifically measured by estimating the incorporation of radio labelled a.Adenine b.Uracil c.Cytosine d.Thymine
  33. 33. • Thymine
  34. 34. 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
  35. 35. • Depend on the action of DNA polymerase
  36. 36. 1.Which of the following is not needed for DNA replication? a. Ribosomes b. DNA c. Nucleotides d. Enzymes
  37. 37. • Ribosomes
  38. 38. • Experimental proof for semiconservative mode of replication is give by A. Watson and Crick B. Meselson & Stahl’s C. Korenberg D. Khurana
  39. 39. • Meselson & Stahl’s
  40. 40. • Idea of DNA replication firstly proposed by A. Watson and Crick B. Crickmore C. Meselson & Stahl’s D. Korenberg
  41. 41. • Watson and Crick
  42. 42. • 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
  43. 43. • Faster in prokaryotes than Eukaryotes
  44. 44. • Which of the following is E. coli origin of replication a. oriA b. oriB c. oriC d. oriD
  45. 45. • oriC
  46. 46. • Which of the following is origin of replication of yeast a. Ori C b. ARS c. ARU d. Ori A
  47. 47. • ARS
  48. 48. • Which of the following protein initiates DNA replication in E.coli a. DnaA b. DnaB c. SSB d. SSA
  49. 49. • DnaA
  50. 50. • DNA polymerase is also known as a. Kornberg's Enzyme b. RNA polymerase c. dNTPs adding and removing enzyme d. PCR enzyme
  51. 51. • Kornberg's Enzyme
  52. 52. 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
  53. 53. • DNA ligase
  54. 54. • 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
  55. 55. • DNA polymerase I

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