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281 lec10 replication_process

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The lecture cover the replication process (mostly in prokaryotes)

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281 lec10 replication_process

  1. 1. Lecture 10: DNA replication in prokaryotes The process of replication Readings (chapter 3) Course 281
  2. 2. Lessons for life
  3. 3. AIMS • Introduce the DNA replication process and the roles of the enzymes/proteins involved. • Introduce the terminology given to the DNA replication fork. This includes: • The strands names and polarity. • The fragments names.
  4. 4. A bit of review What do we need to replicate DNA? 1. DNA template. 2. DNA building blocks (dNTPs). 3. DNA copier enzyme (DNA Polymerase). 4. DNA primer (hook so that the copier know and can start working). 5. Mg2+ so the DNA copier can work. 6. A number of other helpers (proteins and enzymes).
  5. 5. A bit of review DNA Polymerase Exonuclease activity • Nuclease: an enzyme that cuts (digest) nucleotide(s). • Exo: at the ends of a DNA molecule. • Endo: in the middle of a DNA molecule. • Exonuclease: ability to remove nucleotide(s) at the end of a molecule.
  6. 6. A bit of review DNA Polymerase Exonuclease activity • DNA Pol I: • 3’➔5’ exonuclease activity • 5’➔3’ exonuclease activity • DNA Pol III: • 3’➔5’ exonuclease activity
  7. 7. A A bit of review DNA Polymerase 3’➔5’ Exonuclease activity G C T A T T A G C C TG G CA T A C T G T G 5’ 5’ 3’ 3’ A G C TMistake! DNA polymerase goes back (3’ to 5’) and removes wrong nucleotide then adds the correct one (proofreading) G C T A T T A G C C TG G CA T A C T G T G 5’ 5’ 3’ 3’ A G C T DNA template A
  8. 8. A CC A bit of review DNA Polymerase I 5’➔3’ Exonuclease activity G C T This is in its way (5’ to 3’) DNA polymerase I removes forward (5’ to 3’) the nucleotides in its way and adds new ones A G A C T A T T A G C G C A TG G G C T A A T A C T G T G 5’ 5’ 3’ 3’ G C T A G A C T A T T A G C G C TG G G C T A A T A C T G T G 5’ 5’ 3’ 3’
  9. 9. A bit of review For later in the course (Endonuclease) Endonuclease cuts the molecule in the middle G A C T A T T A G C G C A T C G C G G C T A A T T A G C A T C G A T C G 5’ 5’ 3’ 3’ G A C T A T T A G C G C C G C G 5’ 5’ 3’ 3’ A T G C T A A T T A G C A T C G A T C G 5’ 5’ 3’ 3’
  10. 10. DNA synthesis in the cell cycle • The cell cycle involves the replication and synthesis of DNA. • At a specific time in the cell cycle and in the presence of specific conditions, the DNA is replicated. • The DNA is replicated during the S phase of the cell cycle.
  11. 11. Where replication starts? • Initiation of replication start at a specific location: • Replicator. • Origin of replication (Ori). • Origin of replication has specific characteristics: 1. About 245 bp. 2. A-T rich region. Remember: A-T hydrogen bonds! Ori
  12. 12. DNA replication process Ori DnaA • Initiation protein (DnaA) binds to the origin of replication and denatures the double strands forming two single strands of DNA (two templates). 1- DNA denaturation
  13. 13. DNA replication process DnaA • The region after the separation of the two strands is referred to as (the replication bubble). • The strands represent the two template strands needed for replication. • Each side of the replication bubble represent a replication fork. 1- DNA denaturation
  14. 14. DNA replication process • The replication bubble creates two replication forks going to opposite directions. • This is referred to as a bidirectional replication. Replication bubble Two replication forks 1- DNA denaturation/DNA melting
  15. 15. DNA replication process The two forks are identical in the process of replication and for the sake of ease of understanding, we will consider the process on one replication fork. Replication bubble Two replication forks 1- DNA denaturation/DNA melting
  16. 16. DNA replication process DNA helicase (DnaB) is loaded to the fork by the helicase loader (DnaC). 2- Helicase loading 5’3’ 5’ 3’ Helicase (DnaB) Helicase loader (DnaC) Template 2 Template 1
  17. 17. DNA replication process DNA helicase breaks hydrogen bonds between bases and untwist the DNA. 2- Helicase loading 5’3’ 5’ 3’ Helicase (DnaB) Helicase loader (DnaC) Template 2 Template 1
  18. 18. DNA replication process Single strand DNA binding protein binds to ssDNA and prevent the strands from re- annealing (coming back together). 3- Single strand DNA binding proteins(SSB) 5’3’ 5’ 3’ Template 2 Template 1 SSB
  19. 19. DNA replication process DNA primase adds (an RNA primer) at the 5’ providing a 3’-OH for new strand synthesis on both templates. 4- DNA primase 5’3’ 5’ 3’ Helicase (DnaB) Template 2 Template 1 Primase 5’ 3’-H O 5’ 3’-H O
  20. 20. DNA replication process Primase add a single primer on the leading strand and multiple primers on the lagging strand. 4- DNA primase 5’3’ 5’ 3’ Helicase (DnaB) Template 2 Template 1 Primase 5’ 3’-H O 5’ 3’-H O
  21. 21. DNA replication process 5- DNA synthesis by DNA polymerase 5’3’ 5’ 3’ 5’ 3’-H O 3’-H O 5’ DNA Pol III DNA Pol I DNA polymerase extends the primers by adding more nucleotides. DNA polymerase pushes the SSB as it adds more nucleotides.
  22. 22. DNA replication process 5- DNA synthesis by DNA polymerase Remember! DNA has two strands (two templates) that have opposite polarity Template 1: 5’ ➔ 3’ Template 2: 3’ ➔ 5’ DNA polymerase synthesis DNA only 5’➔3’ This makes DNA synthesis continuous on one strand and one discontinuous the other strand
  23. 23. DNA replication process 5- DNA synthesis by DNA polymerase 5’3’ 5’ 3’ 5’ 5’ DNA Pol III DNA Pol I DNA Pol III synthesize DNA continuously because template is always available. DNA Pol I synthesize DNA discontinuously because DNA pol I runs out of template. How to solve this problem?
  24. 24. DNA replication process 5- DNA synthesis by DNA polymerase 5’3’ 5’ 3’ 5’ 5’ DNA Pol I DNA Pol III 2 1 Leading strand template What polarity? Leading strandLagging strand Lagging strand template What polarity?
  25. 25. DNA replication process 5- DNA synthesis by DNA polymerase More primers synthesized as the replication fork moves forward and more template is provided for DNA Pol I
  26. 26. DNA replication process 5- DNA synthesis by DNA polymerase So DNA is replicated in Semi-dis-continuous way
  27. 27. DNA replication process 6- DNA gyrase 5’3’ 5’ 3’ 5’ 5’ DNA Pol I DNA Pol III 2 1 Leading strand template Leading strandLagging strand Lagging strand template
  28. 28. DNA replication process 6- DNA gyrase 3’ As the replication fork, the tension generated by the untwisting DNA of DNA is relaxed by DNA gyrase
  29. 29. DNA replication process 7- Removing primers 5’3’ 5’ 3’ 5’ 5’ DNA Pol I DNA Pol III 2 1 Leading strand template Leading strand Lagging strand Lagging strand template Primer removed
  30. 30. DNA replication process 7- Removing primers DNA pol I on the lagging strand removers the RNA primer using its 5’-3’ exonuclease activity and replaces its location with nucleotides.
  31. 31. DNA replication process 8- Okazaki fragments 5’3’ 5’ 3’ 5’ 5’ DNA Pol III 2 Leading strand template Leading strand Lagging strand Lagging strand template Okazaki fragment
  32. 32. DNA replication process 8- Okazaki fragments • The removal of the primer by DNA pol I generates a nick in the lagging strand where a phosphodiester bond is missing. • Think a about it as a small cut in the strand.
  33. 33. DNA replication process 8- Okazaki fragments sealed by DNA ligase 5’3’ 5’ 3’ 5’ 5’ DNA Pol III 2 Leading strand template Leading strand Lagging strand Lagging strand template Okazaki fragment sealed by DNA Ligase
  34. 34. DNA replication process 8- Okazaki fragments sealed by DNA ligase DNA ligase seals Okazaki fragments on the lagging strand.
  35. 35. The replisome machine For simplicity, we studied every enzyme and the reactions associated to be separate. In reality all units work as a one machine.
  36. 36. The replisome machine All enzymes come together to form a machine called the replisome.
  37. 37. Quiz The replication of the two strands is done in a ……… way a) Continuous b) discontinuous c) semi-continuous d) semi-dis-continuous e) None of the above
  38. 38. To study Endonuclease Exonuclease Cell cycle S phase Replicator Origin of replication Ori Okazaki fragments Lagging strand Leading strand Lagging strand template Lagging strand template DNA polymerase I DNA polymerase III DNA ligase DNA primase primer Gyrase DNA Ligase Replisome DnaA DnaB DnaC SSB Semi-dis-continuous replication Replication fork Replication bubble Helicase DNA melting Denature Reannealing Bidirectional replication
  39. 39. Expectations • You know the process of DNA replication in prokaryotes. • You know the names of the compartments of the replication fork. • You know the sequence of events and where each enzyme/protein function. • You know that all work together and breaking the events into multiple ones is only to make it easier to understand.
  40. 40. For a smile

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