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D. Replication at the Molecular Level

D. Replication at the Molecular Level. D. Replication at the Molecular Level 1. Replication in E. coli

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D. Replication at the Molecular Level

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  1. D. Replication at the Molecular Level

  2. D. Replication at the Molecular Level • 1. Replication in E. coli • a. A specific sequence of bases is recognized as the binding site for the replication complex – this is called the Replication Origin, and the DNA replicated from this site is called a replicon.

  3. D. Replication at the Molecular Level • 1. Replication in E. coli • a. A specific sequence of bases is recognized as the binding site for the replication complex – this is called the Replication Origin, and the DNA replicated from this site is called a replicon. • - Bacteria have only a single replication origin, and the entire circular chromosome is replicated from this point.

  4. 1. Replication in E. coli • a. Replication Origin has 9-mers and 13-mers – homologous sequences… • b. Three enzymes (DNaA,B, C), collectively called “helicases” bind to the origin and break the hydrogen bonds holding the helices together. “Single-strand binding proteins” stabilize the DNA.

  5. 1. Replication in E. coli a. Replication Origin has 9-mers and 13-mers – homologous sequences… b. Three enzymes (DNaA,B, C), collectively called “helicases” bind to the origin and break the hydrogen bonds holding the helices together. “Single-strand binding proteins” stabilize the DNA. c. The enzyme gyrase works downstream, cutting single or double strands to relieved the torque on the molecule. (topoisomerases affect shape).

  6. 1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I.

  7. 1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I.

  8. 1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I. DNA Poly III is the primary replication enzyme, but I-V have repair functions

  9. 1. Replication in E. coli d. DNA Polymerases: And note that NONE can initiate chain synthesis!!!!

  10. 1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I. DNA Poly III is the primary replication enzyme, but I-V have repair functions DNA Poly’s are very complex enzymes with multiple polypeptides and functional groups. And actually, two DNA Poly III enzymes work in parallel as a DIMER

  11. 1. Replication in E. coli d. DNA Polymerases: The DNA Polymerases, the helicases and Gyrase, the Stabilization Proteins, and other enzymes we’ll mention form a HUGE functional replication unit = Replisome

  12. D. DNA Replication at the Molecular Level 1. Replication in E. coli 2. The Process – Replication at the fork: a. Initiation: i . Helicases bind and separate helices, stabilizing protein stabilize the single strands, gyrase relives torque ii. Primase, an RNA Polymerase, begins synthesis of an RNA strand in the 5’3’ direction on both strands: 3’ 5’ 3’

  13. 2. The Process – Replication at the fork: a. Initiation: b. Polymerization: NOW, with a free-3’-OH to add bases to, DNA Polymerase III displaces the Primase and adds DNA bases to extend the new helix in both directions (@50,000 bases/min!):

  14. 2. The Process – Replication at the fork: a. Initiation: b. Polymerization: the B-subunit of DNA Poly III “is a “sliding clamp” that holds the enzymes on the helices:

  15. 2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’).

  16. 2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’). The short sections of discontinuously synthesized DNA are called “Okazaki Fragments”.

  17. 2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’). The short sections of discontinuously synthesized DNA are called “Okazaki Fragments”. This is what is happening at one fork; how about the other?

  18. 5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: The RNA must be removed… 3’ 5’

  19. 5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer… 3’ 5’ 3’ 5’

  20. 5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer…DNA Poly I and II, “fill the gap” with DNA bases, using the free-3’-OH group on the last DNA base to add bases to.... the epsilon subunit is critical in repair… 3’ 5’ 3’ 5’

  21. 5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer…DNA Poly I and II, “fill the gap” with DNA bases, using the free-3’-OH group on the last DNA base to add bases to… then Ligase make the last phosphodiester bond, linking the fragments of DNA together. 3’ 5’

  22. 2. The Process a. Initiation: b. Polymerization: c. Repair: d. Termination: With a circular chromosome (bacteria), it is easy to complete the process.

  23. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes

  24. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication.

  25. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS).

  26. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS). - How are they found by the replisome? - proteins bind to these specific sequences during the G1 phase – these proteins form an “Origin Recognition Complex” (ORC).

  27. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS). - How are they found by the replisome? - proteins bind to these specific sequences during the G1 phase – these proteins form an “Origin Recognition Complex” (ORC). - at the onset of the S phase, protein kinase enzymes bind and form a ‘pre-replication complex’ that binds the DNA Polymerase and helicases and initiates replication. The activated kinases, bound with the replisome, inhibit the formation of more pre-RC, so replication of a region occurs only once.

  28. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: - The ‘core’ DNA bound to histones in a nucleosome must be unwound to be replicated. After this occurs, new histones are made to bind the new double-helices.

  29. D. Replication at the Molecular Level 1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: c. Polymerase Production - base replication rates are 25x slower in eukaryotes - but they have multiple origins and 50,000 x as many Polymerase III enzymes…so the process occurs faster at a cellular level.

  30. 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: c. Polymerase Production d. Telomere Replication - Problem: shortening chromosomes RNA primer

  31. 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: c. Polymerase Production d. Telomere Replication - Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations).

  32. d. Telomere Replication - Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomerase is a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein.

  33. d. Telomere Replication - Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomeraseis a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein. The RNA has several repeats of CCCCAA, which act as a template for the formation of GGGGTT DNA in the process of reverse transcription (reading RNA and making DNA).

  34. d. Telomere Replication - Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomeraseis a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein. The RNA has several repeats of CCCCAA, which act as a template for the formation of GGGGTT DNA in the process of reverse transcription (reading RNA and making DNA). The process continues, forming a long and non-coding platform for primase to begin discontinuous synthesis.

  35. d. Telomere Replication - Problem: shortening chromosomes - Curiously, most mature cells lack telomerase. The shortening of chromosomes may be a signal, or an inhibitor, that prevents further cell division. This is GOOD if it’s a cancer cell; bad otherwise!

  36. IX: DNA Function: Protein Synthesis

  37. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information

  38. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. Why a two-step process? a. Historical contingency… That’s how it evolved from an RNA Protein system…

  39. a. Historical contingency… That’s how it evolved from an RNA Protein system… Conundrum: DNA recipe needs enzymes to be read…. Enzymes are made by reading the DNA recipe… One could not arise before the other, and it is very unlikely that they would arise at the same time by chance.

  40. a. Historical contingency… That’s how it evolved from an RNA Protein system… - in 1979, Thomas Cech discovered a new class of biomolecules – RNA molecules that could catalyze reactions: RIBOZYMES. Because they are RNA, they could act as an information storage molecule AND they were an enzyme-like catalyst. (Nobel Prize) - In the 1990’s, RNA molecules were synthesized that could replicate themselves without enzymes – “self-replicating molecules” Perhaps the first genetic systems were self-replicating RNA molecules… and our modern genetic system evolved from that! How??

  41. Stage 1: Self-replicating RNA evolves RNA

  42. Stage 1: Self-replicating RNA evolves RNA m- , r- , and t- RNA PROTEINS (REPLICATION ENZYMES) Stage 2: RNA molecules interact to produce proteins... if these proteins assist replication (enzymes), then THIS RNA will have a selective (replication/reproductive) advantage... chemical selection.

  43. DNA Reverse transcriptases m- , r- , and t- RNA PROTEINS (REPLICATION ENZYMES) Stage 3: Mutations create new proteins that read RNA and make DNA; existing replication enzymes replicate the DNA and transcribe RNA.

  44. Can this happen? Are their organisms that read RNA and make DNA?

  45. Can this happen? Are their organisms that read RNA and make DNA? yes - retroviruses....

  46. DNA m- , r- , and t- RNA Already have enzymes that can make RNA... PROTEINS (REPLICATION ENZYMES) Stage 3: Mutations create new proteins that read RNA and make DNA; existing replication enzymes replicate the DNA and transcribe RNA.

  47. DNA m- , r- , and t- RNA Already have enzymes that can make RNA... PROTEINS (REPLICATION ENZYMES) Stage 3: Mutations create new proteins that read RNA and make DNA; existing replication enzymes replicate the DNA and transcribe RNA.

  48. This is adaptive because the two-step process is more productive, and DNA is more stable (less prone to mutation). DNA m- , r- , and t- RNA PROTEINS (REPLICATION ENZYMES) Stage 4: Mutations create new proteins that replicate the DNA instead of replicating the RNA...

  49. This is adaptive because the two-step process is more productive, and DNA is more stable (less prone to mutation). DNA And that's the system we have today.... m- , r- , and t- RNA PROTEINS (REPLICATION ENZYMES) Stage 4: Mutations create new proteins that replicate the DNA instead of replicating the RNA...

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