DNA Replication and Recombination: Eukaryotic Mechanisms and Meiotic Context

DNA Replication and Recombination

Lecture Objectives

• Discuss eukaryotic DNA replication mechanisms.

• Explain telomere structure and the "end replication problem."

• Understand DNA recombination in the context of meiosis.

DNA as a Template

Template Function of DNA

DNA serves as a template for its own replication due to the specific arrangement and nature of its nitrogenous bases. The complementarity of DNA strands allows each strand to serve as a template for synthesis of the other.

• Complementarity: Adenine (A) pairs with Thymine (T), and Guanine (G) pairs with Cytosine (C).

• Template Mechanism: Each strand guides the formation of a new complementary strand during replication.

• Example: If the template strand is 5'-ATGC-3', the new strand will be 3'-TACG-5'.

Models of DNA Replication

Three Possible Models

Early hypotheses proposed three models for DNA replication:

• Conservative Model: The original double helix remains intact, and a completely new double helix is synthesized.

• Semiconservative Model: Each new DNA molecule consists of one old (parental) strand and one newly synthesized strand.

• Dispersive Model: Parental DNA is dispersed throughout both strands of the two daughter helices.

Diagram: (Described) The conservative model keeps the parental helix intact; the semiconservative model splits the parental strands between two daughter helices; the dispersive model mixes parental and new DNA in both strands.

Meselson-Stahl Experiment (1958)

Experimental Evidence for Semiconservative Replication

The Meselson-Stahl experiment used 15N-labeled E. coli grown in medium containing heavy nitrogen to distinguish old and new DNA strands.

• Method: Cells were grown in 15N medium, then transferred to 14N medium. DNA was extracted after each generation and analyzed by density gradient centrifugation.

• Result: Each new DNA molecule consisted of one old and one newly synthesized strand, supporting the semiconservative model.

• Significance: Provided strong evidence that DNA replication in prokaryotes is semiconservative.

Overview of DNA Replication

Key Steps in Eukaryotic DNA Replication

DNA replication is a highly regulated, multistep process involving several enzymes and proteins.

1. Unwind: The DNA double helix is unwound.

2. Initiate Elongation: Elongation is initiated by synthesis of an RNA primer.

3. Synthesize: Daughter DNA strands are synthesized by DNA polymerases.

4. Seal: Newly synthesized DNA fragments are sealed together.

• Leading Strand: Synthesized continuously in the 5' to 3' direction.

• Lagging Strand: Synthesized discontinuously as Okazaki fragments (~200 nucleotides in eukaryotes), which are later joined by DNA ligase.

Step 1: Unwind

DNA Helicase and Stabilization

Unwinding of the DNA double helix is essential for replication and is accomplished by several proteins:

• DNA Helicase: Enzyme composed of DnaB polypeptides; unwinds the double helix by breaking hydrogen bonds between bases. Requires energy from ATP hydrolysis.

• Single-Stranded Binding Proteins (SSBPs): Bind to exposed single-stranded DNA to stabilize it and prevent reannealing.

• DNA Gyrase: A type of DNA topoisomerase that relieves supercoiling tension generated by unwinding. Makes single- or double-stranded cuts and is driven by ATP hydrolysis.

Step 2: Initiate Elongation

RNA Primers and DNA Polymerase α

DNA synthesis cannot begin without a short RNA primer, which provides a free 3'-OH group for DNA polymerases to extend.

• DNA Primase: A complex of four proteins; p48 and p58 form the primase (an RNA polymerase), while p68 and p180 form DNA polymerase α.

• Function: Primase synthesizes a short RNA primer (8–12 nucleotides).

• DNA Polymerase α: Extends the RNA primer with ~20 nucleotides of DNA.

• Importance: The free 3'-OH is required for DNA polymerases δ and ε to continue elongation.

Step 3: Elongate

DNA Polymerases and Strand Synthesis

Elongation of DNA is performed by several DNA polymerases:

• DNA Polymerases α, δ, and ε: All involved in nuclear genome replication.

• Polymerase α: Initiates synthesis with primase; possesses low processivity.

• Polymerase δ: Synthesizes the lagging strand during elongation.

• Polymerase ε: Synthesizes the leading strand during elongation.

Polymerase Switching

• Once the RNA primer is in place, polymerase α is replaced by polymerases δ and ε for elongation.

Proliferating Cell Nuclear Antigen (PCNA)

• PCNA: A sliding clamp that increases the processivity of polymerase δ during elongation.

Nucleotide Addition Mechanism

• DNA polymerase adds nucleotides one at a time to the 3' end of the growing strand.

• Each addition involves cleavage of two terminal phosphates, exposing a new 3'-OH group for further extension.

Equation:

$(dNTP) + (DNA_{(n)}) \rightarrow (DNA_{(n+1)}) + PP_i$

Base Pairing and Complementarity

• Adenine pairs with Thymine; Guanine pairs with Cytosine.

• Bond strength: GC pairs (three hydrogen bonds) are stronger than AT pairs (two hydrogen bonds).

Proofreading and Error Correction

• DNA polymerase possesses 3'→5' exonuclease activity for proofreading.

• Incorrectly paired bases are excised and replaced, increasing fidelity of replication.

Discontinuous Synthesis and Okazaki Fragments

• DNA polymerase can only add nucleotides to the 3'-OH end.

• Lagging strand is synthesized discontinuously as Okazaki fragments, which are later joined by DNA ligase.

Removal of RNA Primers

• DNA polymerase δ works with Flap Endonuclease I (FEN1) to remove RNA primers from the lagging strand.

• FEN1 removes RNA:DNA hybrids, allowing replacement with DNA.

Summary Table: Key Enzymes in Eukaryotic DNA Replication

Enzyme/Protein	Function
DNA Helicase	Unwinds the DNA double helix
SSBPs	Stabilize single-stranded DNA
DNA Gyrase	Relieves supercoiling tension
DNA Primase	Synthesizes RNA primer
DNA Polymerase α	Extends RNA primer with DNA
DNA Polymerase δ	Synthesizes lagging strand
DNA Polymerase ε	Synthesizes leading strand
PCNA	Sliding clamp for processivity
FEN1	Removes RNA primers
DNA Ligase	Seals Okazaki fragments

*Additional info: Later slides (not shown here) would cover telomere structure, the end replication problem, and DNA recombination in meiosis. These topics are essential for understanding chromosome stability and genetic diversity in eukaryotes.*