Lec 30 Flashcards - Prokaryotic DNA Replication

Objectives of Prokaryotic DNA Replication

• Understand the three phases of DNA replication: Initiation, Elongation, Termination.

• Learn about bidirectional replication and the origin of replication (OriC).

• Identify the roles of replication fork proteins, RNA primers, and Okazaki fragments on leading and lagging strands.

• Differentiate between E. coli's three major DNA polymerases: Pol I, Pol II, and Pol III.

• Comprehend the function of clamp and clamp loader proteins in DNA synthesis.

• Recognize the role of DNA methylation in distinguishing between original and newly synthesized DNA.

• Grasp the process of DNA replication termination in prokaryotes.

What is DNA Replication?

• Definition: DNA replication is the process of copying a cell's entire genome using an existing strand as a template. It involves complex molecular events and is essential for cell division.

• Stages of DNA Replication:

  1. Initiation: The start of DNA replication.

  2. Elongation: The actual synthesis of new DNA strands.

  3. Termination: The completion and separation of newly synthesized DNA strands.

DNA Replication Dynamics

• Bidirectional Replication:

  • Forms a "Replication Bubble" with two replication forks.

  • The top strand is copied in one direction, while the bottom strand is copied in the opposite direction.

Semiconservative DNA Replication

• Mechanisms:

  1. Conservative: Both original strands remain together.

  2. Dispersive: Pieces of old and new strands are mixed.

  3. Semiconservative: Each daughter cell receives one original and one new strand.

• Proof of Semiconservative Replication: Demonstrated by Mendelson and Stahl's studies using radioactive nitrogen.

Characteristics of Prokaryotic DNA Replication

• Organism Example: E. coli.

• Genome size: ~4 million base pairs, circular structure.

• Replication is semiconservative: each daughter chromosome contains one parental and one new strand.

Key Steps in Initiation of DNA Replication in E. coli

1. OriC Recognition:

   • DnaA (initiator protein) binds to OriC, bending the DNA.

2. Unwinding by DnaB:

   • DnaB (helicase) separates the parental strands and opens the replication bubble.

3. Role of Gyrase:

   • Type II Topoisomerase that unwinds DNA ahead of the fork.

4. RNA Primer Synthesis:

   • DNA primase generates an RNA primer necessary for DNA polymerase to synthesize DNA.

5. Formation of Replisome Complex:

   • Includes DNA polymerase and other proteins necessary for replication.

Leading and Lagging Strands

• Leading Strand: Synthesized continuously towards the replication fork with one primer.

• Lagging Strand: Synthesized discontinuously away from the fork in short fragments (Okazaki fragments) with multiple primers.

Okazaki Fragments and Gaps Filling

• Synthesis: Short DNA segments made by DNA Pol III on the lagging strand.

• Filling Gaps:

  1. RNase H removes RNA primers.

  2. DNA Pol I replaces RNA with DNA.

  3. DNA Ligase joins the fragments together. This process ensures the creation of a continuous DNA strand, essential for maintaining the integrity of the genetic information during cell division.

Prokaryotic DNA Polymerases

Include DNA Pol I, II, and III, each having distinct roles in DNA replication and repair.

DNA Pol I - is responsible for replacing RNA primers,

DNA Pol III - is the primary enzyme for synthesizing new DNA strands during replication.

DNA Pol II - plays a significant role in repairing damaged DNA.

Elongation by DNA Polymerase III

• Processivity: Pol III can add multiple nucleotides without dissociating.

  • This high processivity is crucial for efficient DNA synthesis, allowing the enzyme to rapidly extend the growing DNA strand. Additionally, Pol III's interaction with the sliding clamp, known as the beta-clamp, further enhances its ability to remain attached to the DNA template during elongation. This interaction minimizes the likelihood of errors during replication, ensuring high fidelity in the newly synthesized DNA.

• Exonuclease Activity: 3' to 5' for proofreading to correct mistakes.

• Error Rate: Approximately 1 error per 10^6 nucleotides.

parental DNA is always read in the 3 - 5 direction (leading stand)

Nucleotides are always added to the new strand in the 5-3 direction.

Termination of DNA Replication in E. coli

• Catenation: Two circular DNA strands linked together post-replication; need to separate.

• Role of Topoisomerases: Break and seal strands to separate daughter chromosomes.

• Termination Sites (Ter): Bound by TUS proteins which stop helicase activity.

Summary of Prokaryotic DNA Replication Steps

1. Initiation: OriC binding, helicase action, RNA primer synthesis.

2. Elongation: Continuous leading strand synthesis; lagging strand made of Okazaki fragments, with RNA primers replaced and gaps filled.

3. Termination: TUS proteins bind Ter sites, allowing for separation of DNA.

Key Points to Remember

• Replication is bidirectional and semiconservative; synthesis occurs in a 5' to 3' direction.

• RNA primers are essential for starting DNA replication.

• Leading strand is continuous, lagging strand is discontinuous (Okazaki fragments).

• Proofreading is performed by DNA polymerase III’s exonuclease activity to maintain fidelity.

• RNase H and DNA Pol I work together to replace RNA primers, and DNA Ligase seals the gaps.

What happens as the Replication Fork is formed?

1) Double-standed dsDNA is separated into single-stranded ssDNA, by the DNA Helicase enzyme, at the orgin ORIC.

2) Gyrases, out front of each fork unwind, the supercoiled DNA so it can be replicated.

3) The RNA primer is synthesized by the DNA Primase enzyme.

4) DNA Polymerase III begins adding complementary DNA nucleotides to the 3' end of the RNA primer, elongating the new DNA strand in a 5' to 3' direction.

5) As replication progresses, DNA Polymerase I removes the RNA primers and replaces them with DNA nucleotides to ensure the entire strand is composed of DNA. 6) Finally, the DNA Ligase enzyme seals any nicks in the sugar-phosphate backbone, completing the formation of a continuous double-stranded DNA molecule.

The Trombone model - a dynamic visualization of the DNA replication process, illustrates the coordinated movements of the replication machinery at the replication fork, allowing for efficient synthesis of both leading and lagging strands. This model highlights how the lagging strand is synthesized in short Okazaki fragments, which are later joined together by DNA ligase, ensuring both strands are synthesized accurately and efficiently.

Major enzymes involved in Prokaryotic DNA Replications:

• DNA Polymerase III: The primary enzyme responsible for synthesizing new DNA strands by adding nucleotides to the growing DNA chain.

• Helicase: Unwinds the double helix at the replication fork, separating the two strands to allow access for replication.

• Primase: Synthesizes short RNA primers that provide a starting point for DNA synthesis by DNA Polymerase III.

• Ligase: Joins Okazaki fragments on the lagging strand, sealing the nicks in the sugar-phosphate backbone to create a continuous DNA strand.

• Single-Stranded Binding Proteins (SSBs): Bind to the separated DNA strands, preventing them from re-annealing and protecting them from degradation during the replication process.

• DNA Polymerase III: The main enzyme responsible for synthesizing new DNA strands by adding nucleotides complementary to the template strand, operating in a 5' to 3' direction.

• DNA polymerase II: Plays a role in DNA repair and is involved in the proofreading of newly synthesized DNA to ensure accuracy.

• DNA polymerase I: Responsible for removing RNA primers used during DNA replication and replacing them with DNA nucleotides, thus completing the synthesis of the lagging strand.

• RNase H: An enzyme that removes RNA primers from the RNA-DNA hybrid during DNA replication, facilitating the subsequent replacement of these segments by DNA polymerase I.

• Gyrases: enzymes that relieve the strain generated ahead of the replication fork by introducing negative supercoils into the DNA, crucial for preventing overwinding during the replication process.

• Replisome complex: A multi-protein complex that oversees the replication of DNA; it is composed of several key components, including DNA polymerases, helicases, and other associated factors that work together to ensure accurate and efficient DNA synthesis.

• TUS proteins: Proteins that bind to specific sites on the DNA, preventing the movement of the replication fork and ensuring that replication terminates properly.

• Topoisomerases: Enzymes that manage DNA supercoiling by cutting, twisting, and rejoining DNA strands, allowing for the proper unwinding and separation of the double helix during replication.

three types of Semiconservative DNA Replication:

• Conservative replication: The original DNA molecule remains intact, while an entirely new double helix is formed.

• Dispersive replication: The existing DNA strands are broken into fragments, and new DNA is synthesized in a way that incorporates both old and new material throughout the strands.

• Semiconservative replication: Each new DNA molecule consists of one original strand and one newly synthesized strand, ensuring genetic continuity.