![Origin of replication
• In prokaryotic organisms, DNA replication begins at a single, unique nucleotide
sequence—a site called the origin of replication
• [Note: This is referred to as a consensus sequence, because the order of
nucleotides is essentially the same at each site.]
• This site includes a short sequence composed almost exclusively of AT base pairs
that facilitate melting.
• In eukaryotes, replication begins at multiple sites along the DNA helix
• Having multiple origins of replication provides a mechanism for rapidly replicating
the great length of the eukaryotic DNA molecules.](https://image.slidesharecdn.com/2-200426151436/85/Replication-fork-in-prokaryotic-replication-6-320.jpg)
![b. DNA helicases:
• bind to ssDNA near the replication fork
• then move into the neighboring double stranded region, forcing the
strands apart—in effect, unwinding the double helix.
• require energy provided by ATP
• [Note: DnaB is the principal helicase of replication in E. coli. Its binding to
DNA requires DnaC.]](https://image.slidesharecdn.com/2-200426151436/85/Replication-fork-in-prokaryotic-replication-11-320.jpg)
![2. Solving the problem of supercoils
• As the two strands of the double helix are separated,
• a problem is encountered, namely, the appearance of positive supercoils (also called
supertwists) in the region of DNA ahead of the replication fork (Figure 29.11).
• The accumulating positive supercoils interfere with further
unwinding of the double helix.
• [Note: Supercoiling can be demonstrated by tightly grasping one end of a helical
telephone cord while twisting the other end. If the cord is twisted in the direction of
tightening the coils, the cord will wrap around itself in space to form positive
supercoils. If the cord is twisted in the direction of loosening the coils, the cord will
wrap around itself in the opposite direction to form negative supercoils.]
• To solve this problem, there is a group of enzymes called DNA
topoisomerases, which are responsible for removing supercoils in
the helix.](https://image.slidesharecdn.com/2-200426151436/85/Replication-fork-in-prokaryotic-replication-18-320.jpg)
Replication fork in prokaryotic replication
- 1. III. STEPS IN PROKARYOTIC DNA SYNTHESIS Lecture 2 A. Separation of the two complementary DNA strands B. Formation of the replication fork C. Direction of DNA replication D. RNA primer E. Chain elongation F. Excision of RNA primers and their replacement by DNA
- 2. • When the two strands of the DNA double helix are separated, each can serve as a template for the replication of a new complementary strand. • This produces two daughter molecules, each of which contains two DNA strands with an antiparallel orientation • This process is called semiconservative replication because, although the parental duplex is separated into two halves (and, therefore, is not “conserved” as an entity), each of the individual parental strands remains intact in one of the two new duplexes (Figure 29.8).
- 3. • The enzymes involved in the DNA replication process are template- directed polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. • In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated
- 5. A. Separation of the two complementary DNA strands • In order for the two strands of the parental double helical DNA to be replicated, they must first separate (or “melt”) over a small region, because the polymerases use only ssDNA as a template.
- 6. Origin of replication • In prokaryotic organisms, DNA replication begins at a single, unique nucleotide sequence—a site called the origin of replication • [Note: This is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.] • This site includes a short sequence composed almost exclusively of AT base pairs that facilitate melting. • In eukaryotes, replication begins at multiple sites along the DNA helix • Having multiple origins of replication provides a mechanism for rapidly replicating the great length of the eukaryotic DNA molecules.
- 8. B. Formation of the replication fork • As the two strands unwind and separate, they form a “V” where active synthesis occurs. This region is called the replication fork. • It moves along the DNA molecule as synthesis occurs. • Replication of dsDNA is bidirectional—that is, the replication forks move in opposite directions from the origin, generating a replication bubble.
- 9. 1. Proteins required for DNA strand separation • Initiation of DNA replication requires the recognition of the origin of replication by a group of proteins that form the prepriming complex. • These proteins are responsible for maintaining the separation of the parental strands, and for unwinding the double helix ahead of the advancing replication fork. • DnaA protein • DNA helicases • Single-stranded DNA-binding (SSB) proteins • DNA polymerase
- 10. a. DnaA protein: • binds to specific nucleotide sequences at the origin of replication • causing short, tandemly arranged (one after the other) AT-rich regions in the origin to melt. • Melting is ATP-dependent • results in strand separation with the formation of localized regions of ssDNA.
- 11. b. DNA helicases: • bind to ssDNA near the replication fork • then move into the neighboring double stranded region, forcing the strands apart—in effect, unwinding the double helix. • require energy provided by ATP • [Note: DnaB is the principal helicase of replication in E. coli. Its binding to DNA requires DnaC.]
- 12. c. Single-stranded DNA-binding (SSB) proteins: • bind to the ssDNA generated by helicases • bind cooperatively—that is, the binding of one molecule of SSB protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand. • are not enzymes, but rather serve to shift the equilibrium between dsDNA and ssDNA in the direction of the single- stranded forms. Functions 1. keep the two strands of DNA separated in the area of the replication origin, 2. Provide the single-stranded template required by polymerases, but also protect the DNA from nucleases that degrade ssDNA
- 13. d. DNA polymerase - the enzymes that make DNA 1. Pol I • It has three distinct functions 1. polymerization 2. 3’→5’ exonuclease 3. 5’→3’ exonuclease • Its structure has 2 domains • Large domain (Klenow fragment) • Small domain Three DNA Polymerases in E. coli
- 15. • 2. Pol II • It has the polymerization and 3’ → 5’ exonuclease activity • 3. Pol III • The pol III core is composed of three subunits, α, ε, and θ. • The α-subunit has the DNA polymerase activity. • The ε-subunit has the 3’→5’ exonuclease activity that carries out proofreading. • The role of the θ -subunit is not yet clear. • β-sliding clamp • ϒ- clamp loader - Sliding clamp • *
- 16. So, DNA polymerase 1. can not synthesize on it own 2. Ability for 5’→3’ polymerization 3. needs something on which it can jump on that is called primer • Primer can not be DNA • 10-12 nucleotides • Always in the form of RNA 4. Unable to ligate the DNA
- 17. DNA polymerase Properties Pol I Pol II Pol III Repair phenomena x Major Replication Polymerization 5’→3’ + + + Exonuclease 3’→5’ + + + 5’→3’ + - -
- 18. 2. Solving the problem of supercoils • As the two strands of the double helix are separated, • a problem is encountered, namely, the appearance of positive supercoils (also called supertwists) in the region of DNA ahead of the replication fork (Figure 29.11). • The accumulating positive supercoils interfere with further unwinding of the double helix. • [Note: Supercoiling can be demonstrated by tightly grasping one end of a helical telephone cord while twisting the other end. If the cord is twisted in the direction of tightening the coils, the cord will wrap around itself in space to form positive supercoils. If the cord is twisted in the direction of loosening the coils, the cord will wrap around itself in the opposite direction to form negative supercoils.] • To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix.
- 19. a. Type I DNA topoisomerases: • enzymes reversibly cut one strand of the double helix • have both nuclease (strand-cutting) and ligase (strand-resealing) activities • do not require ATP, but rather appear to store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand (Figure 29.12) • Each time a transient “nick” is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving (“relaxing”) accumulated supercoils. • Enzyme relax negative supercoils in E. coli, and both negative and positive supercoils in eukaryotic cells.
- 20. b. Type II DNA topoisomerases: • enzymes bind tightly to the DNA double helix and make transient breaks in both strands. • enzyme then causes a second stretch of the DNA double helix to pass through the break and, finally, reseals the break • As a result, both negative and positive supercoils can be relieved by this ATP-requiring process. • are also required in both prokaryotes and eukaryotes for the separation of interlocked molecules of DNA following chromosomal replication.
- 21. • DNA gyrase, a Type II topoisomerase found in bacteria and plants, has the unusual property of being able to introduce negative supercoils into relaxed circular DNA using energy from the hydrolysis of ATP. • This facilitates the future replication of DNA because the negative supercoils neutralize the positive supercoils introduced during opening of the double helix. It also aids in the transient strand separation required during transcription.
- 22. Topoisomerase I Topoisomerase II monomeric Tetrameric No ATP ATP required 1 gene, topI 2 gene, gyrase I & II Cuts one of the two treads Cut both threads 100 KDa 400 KDa Use Mg+2 Do not use Mg+2
- 24. • Explain how the complimentary strands of DNA will separate for replication • Describe all the proteins/enzymes involved for this purpose HOW TO SUBMIT THE HAND WRITTEN ASSIGNMENT 1. Take a neat paper and a pen. 2. First of all, Write your complete name and class 3. Then Start the discussion of your answer 4. Try to write in points and then explain those points wisely 5. When you are done, take pictures of all the answer sheets by the clean scan Application (https://play.google.com/store/apps/details?id=com.indymobileapp.document.scanner )or Tap Scanner App ( https://play.google.com/store/apps/details?id=pdf.tap.scanner ) 6. The pictures will be saved in pdf format by default. Send your assignment in pdf format to this address. anamtariq77@gmail.com
- 25. The End