أسئلة المحاضرة الرابعة جينتكس (بعد التعديل)

DNA Replication

• DNA replication is the process of copying a DNA molecule into an identical copy.
• DNA replication occurs before cell division, so that the two daughter cells have identical DNA copies.

Semiconservative Replication

• Semiconservative replication refers to the mechanism by which each of the two resulting double-stranded DNA molecules contains one old strand and one newly synthesized strand from the original molecule.
• One old strand (parent strand) is conserved.
• One new strand is synthesized using free nucleotides found in the nucleus.
• Both strands serve as a template for DNA replication simultaneously.

Criteria for DNA Replication

• Replication is bidirectional.
• DNA synthesis occurs in the 5' to 3' direction.
• DNA replication requires a complex of enzymes and proteins.

Components of DNA Replication

• Template: Double-stranded DNA.
• Precursors: dATP, dGTP, dCTP, dTTP (deoxyribonucleotide triphosphates)
• Cofactors: Mg++, Mn++, ATP.
• Enzymes: DNA Polymerases, DNA helicases, DNA primase (a subunit of DNA polymerase α), Exonucleases.
• Proteins: Single-stranded DNA binding proteins (ssDB Proteins), Nick sealing enzyme DNA ligase.

DNA Polymerase

• DNA polymerase synthesizes a new DNA strand using the template strand.
• It reads the template strand in the 3' to 5' direction and synthesizes the new strand in the 5' to 3' direction.
• Deoxyribonucleotide triphosphates (dATP, dGTP, dCTP & dTMP) serve as precursors for the new strand.
• DNA polymerase cannot initiate DNA synthesis and it needs an RNA primer.
• There are five types of DNA polymerases in eukaryotes: α, ε, β, γ and δ.

Initiation of DNA Replication

• DNA replication begins at multiple DNA sites called origins of replication (ori).
• Origins of replication are rich in AT base pairs (consensus sequence).
• Multiple origins of replication begin simultaneously so replication occurs bidirectionally.
• DNA helicases break the hydrogen bonds between the two DNA strands, unwinding the double helix and creating a "replication bubble."
• Replication forks - The V-shaped structure at each end of the replication bubble.
• Single-stranded binding proteins (ssBP) hold the separated DNA strands apart to prevent them from rejoining.

Priming by Primase

• Primase is an RNA polymerase.
• Primase synthesizes a short RNA primer that provides a starting point for DNA polymerase.
• One RNA primer is required for the leading strand.
• Multiple primers are needed for the lagging strand.

Synthesis of Leading and Lagging Strands

• DNA polymerases are responsible for synthesizing both strands of DNA.
• The presence of many DNA polymerases (more than 20,000) reduces the time needed for replication.

Leading Strand

• Synthesized continuously.
• Replicated by DNA polymerase ε in the direction of the advancing replication fork.

Lagging Strand

• Synthesized discontinuously.
• Replicated by DNA polymerase δ in the opposite direction of the advancing replication fork.
• Synthesized in fragments called Okazaki fragments.

Excision of RNA Primers

• RNA primers are removed by exonucleases.

Ligation of DNA Fragments

• The final phosphodiester linkage between the 5'-phosphate group on one DNA chain and the 3'-hydroxyl group on the adjacent chain is catalyzed by DNA ligase.
• This reaction requires ATP.

Proofreading of Newly Synthesized DNA Strands

• DNA polymerases δ and ε have proofreading activity.
• They check for errors in base pairing and correct them, ensuring the accuracy of DNA replication.

DNA Replication

• DNA replication is the process of creating an identical copy of a DNA molecule.
• It occurs before cell division (mitosis) ensuring each daughter cell receives a complete set of DNA.
• DNA replication is semiconservative, meaning each new DNA molecule contains one original strand (parent strand) and one newly synthesized strand.
• Both DNA strands serve as templates for replication simultaneously.
• Replication proceeds bidirectionally, meaning it happens in both directions from the origin of replication.
• The direction of new DNA synthesis is always 5' to 3'.

Key Components of DNA Replication

• DNA Polymerases: The main enzymes responsible for synthesizing new DNA strands.
  • They require an existing RNA primer to begin replication.
  • In eukaryotes, there are five DNA polymerases: α, ε, β, γ, and δ.
• DNA Helicase: Unwinds the double helix structure of DNA, separating the two strands.
• Single-stranded DNA binding proteins (ssBPs): Prevent the separated strands from re-annealing, keeping them accessible for replication.
• DNA Primase: Synthesizes short RNA primers that provide a starting point for DNA polymerase to attach to.
• Exonucleases: Remove RNA primers from the newly synthesized DNA strands.
• DNA Ligase: Seals the gaps between newly synthesized DNA fragments, creating a continuous strand.

Stages of DNA Replication

• Initiation: Begins at specific sites called "origins of replication" (ori)
  • Ori regions are rich in AT base pairs (adenine and thymine) due to their weaker bonding, making them easier to separate.
• Unwinding and Separation:
  • DNA helicase unwinds the double helix at the origin of replication, creating a "replication bubble".
  • Two replication forks form at the ends of the bubble, and replication proceeds in opposite directions.
  • Single-stranded binding proteins (ssBPs) prevent the separated strands from re-annealing.
• Priming:
  • Primase creates short RNA primers on both strands, providing starting points for DNA polymerase.
  • The leading strand only needs one primer, while the lagging strand needs multiple primers.
• Synthesis of Leading and Lagging Strands:
  • Leading strand: Synthesized continuously in the 5' to 3' direction, following the replication fork movement.
  • Lagging strand: Synthesized discontinuously in fragments (Okazaki fragments) in the opposite direction of fork movement.
• Excision of RNA Primers:
  • Exonucleases remove the RNA primers.
• Ligation:
  • DNA ligase joins the Okazaki fragments on the lagging strand and fills in any remaining gaps to make a continuous strand.

Proofreading Mechanism

• DNA polymerases (δ and ε) have a proofreading function.
• This mechanism helps ensure that the newly synthesized DNA strand is accurate and free of errors.
• DNA polymerase can backtrack (3' to 5' direction) and remove mismatched nucleotides.