DNA and the Gene: Synthesis and Repair – Study Notes

DNA and the Gene: Synthesis and Repair

Testing Early Hypotheses about DNA Synthesis

Understanding how DNA replicates was a foundational question in molecular biology. Three main models were proposed:

• Semiconservative replication: Parental DNA strands separate and each serves as a template for a new daughter strand. Each daughter DNA molecule consists of one old and one new strand.

• Conservative replication: The parental DNA molecule serves as a template for an entirely new molecule, so one daughter molecule is all old DNA and the other is all new DNA.

• Dispersive replication: The parent DNA is cut into small pieces, and each daughter molecule contains interspersed segments of old and new DNA.

Example: The Meselson–Stahl experiment provided evidence for the semiconservative model by using isotopic labeling to distinguish old and new DNA strands.

A Model for DNA Synthesis

DNA Polymerase and Directionality

DNA polymerase is the enzyme responsible for catalyzing DNA synthesis. Several types exist, but all share key properties:

• They work only in one direction: new nucleotides are added to the 3' end of a growing DNA chain, so synthesis proceeds in the 5' → 3' direction.

• DNA polymerization is endergonic, but the energy comes from the high-energy bonds in deoxyribonucleoside triphosphates (dNTPs).

• Formation of phosphodiester bonds during DNA synthesis is exergonic due to the hydrolysis of dNTPs.

Equation:

$\text{(dNMP)}_n + \text{dNTP} \rightarrow \text{(dNMP)}_{n+1} + \text{PP}_i$

Origin of Replication and Replication Bubbles

DNA replication begins at specific sequences called origins of replication:

• Bacteria typically have a single origin per chromosome, forming one replication bubble.

• Eukaryotic chromosomes have multiple origins, forming many replication bubbles.

• Each bubble has two replication forks where DNA is actively synthesized in both directions (bidirectional replication).

Opening and Stabilizing the DNA Helix

Several proteins are required to open and stabilize the DNA double helix:

• DNA helicase: Breaks hydrogen bonds between DNA strands, separating them.

• Single-strand DNA-binding proteins (SSBPs): Bind to separated strands to prevent them from re-annealing.

• Topoisomerase: Relieves tension created by unwinding the helix by cutting and rejoining DNA downstream of the replication fork.

Leading Strand Synthesis

The antiparallel structure of DNA means that synthesis occurs differently on each strand. The leading strand is synthesized continuously toward the replication fork:

• DNA polymerase cannot start synthesis de novo; it requires a free 3' hydroxyl group.

• A short RNA primer, synthesized by primase (an RNA polymerase), provides this 3' end.

• DNA polymerase then adds dNTPs to the primer’s 3' end, synthesizing the new DNA strand in the 5' → 3' direction.

Lagging Strand Synthesis

The lagging strand is synthesized discontinuously, away from the replication fork, as short fragments called Okazaki fragments:

• Primase synthesizes new RNA primers as the replication fork opens.

• DNA polymerase synthesizes short DNA fragments from these primers.

• Fragments are later joined into a continuous strand by DNA ligase.

Proteins Required for DNA Synthesis in Bacteria

The following table summarizes the main proteins involved in bacterial DNA replication:

Enzyme/Protein	Function
Helicase	Catalyzes the separation of DNA strands to open the double helix
Single-strand DNA-binding proteins (SSBPs)	Stabilize single-stranded DNA
Topoisomerase	Relieves twisting forces caused by the opening of the helix
Primase	Catalyzes the synthesis of RNA primer
DNA polymerase III	Extends the leading strand and Okazaki fragments
Sliding clamp	Holds DNA polymerase in place during strand extension
DNA polymerase I	Removes RNA primer and replaces it with DNA
DNA ligase	Joins Okazaki fragments into a continuous strand

Replicating the Ends of Linear Chromosomes

The End Replication Problem and Telomeres

Replication of linear eukaryotic chromosomes presents a unique challenge at the ends, called telomeres:

• Leading strand synthesis can proceed to the very end, but lagging strand synthesis leaves a single-stranded overhang after primer removal.

• This overhang is eventually degraded, causing chromosomes to shorten with each cell division.

• Telomeres consist of short, repeating, non-coding DNA sequences that protect genes from erosion.

Telomerase and Chromosome Maintenance

Telomerase is an enzyme that extends telomeres, solving the end replication problem:

• Telomerase carries its own RNA template and adds DNA repeats to the overhang.

• Once the overhang is long enough, normal DNA polymerase can complete lagging strand synthesis.

• Telomerase is active in gametes and stem cells, but not in most somatic cells.

Example: Most cancer cells reactivate telomerase, allowing unlimited cell division.

Correcting Mistakes in DNA Synthesis

DNA Polymerase Proofreading

DNA polymerase has a proofreading function to ensure high fidelity during replication:

• Incorrectly paired bases are detected by their abnormal shape.

• The enzyme’s exonuclease site removes the mismatched nucleotide.

• Proofreading reduces the error rate to about one mistake per 107 nucleotides.

Mismatch Repair

Sometimes, mismatches escape proofreading. Mismatch repair enzymes correct these errors after DNA synthesis:

• They recognize the mismatch, remove a segment of the newly synthesized strand, and fill in the correct bases using the template strand.

Repairing Damaged DNA

DNA can be damaged by environmental factors such as UV light, X-rays, and chemicals:

• UV light can cause thymine dimers, which create kinks in the DNA and block replication.

• The nucleotide excision repair system recognizes distortions, removes the damaged DNA, and fills in the gap using the undamaged strand as a template.

• DNA ligase seals the repaired strand into the backbone.

Summary Table: Key Steps and Enzymes in DNA Replication and Repair

Process	Key Enzyme(s)	Function
Initiation	Helicase, SSBPs, Topoisomerase	Unwind and stabilize DNA
Primer synthesis	Primase	Synthesize RNA primer
Elongation	DNA polymerase III, Sliding clamp	Synthesize new DNA strand
Primer removal	DNA polymerase I	Replace RNA primer with DNA
Ligation	DNA ligase	Join DNA fragments
Proofreading	DNA polymerase	Remove mismatched bases
Mismatch repair	Mismatch repair enzymes	Correct errors post-replication
Damage repair	Nucleotide excision repair system	Remove and replace damaged DNA
Telomere extension	Telomerase	Extend chromosome ends

Additional info: The accuracy and efficiency of DNA replication and repair are essential for genetic stability and prevention of diseases such as cancer.