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, preserving the original molecule intact.

• 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 the Direction of Synthesis

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

• They work only in one direction, adding deoxyribonucleotides to the 3' end of a growing DNA chain.

• DNA synthesis always proceeds in the 5' to 3' direction.

DNA polymerization is energetically favorable because the monomers, deoxyribonucleoside triphosphates (dNTPs), have high potential energy due to their three phosphate groups. Hydrolysis of these phosphates drives the formation of phosphodiester bonds, making the reaction exergonic.

Origin of Replication and Replication Bubbles

DNA replication begins at specific sequences called origins of replication:

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

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

• Each bubble contains two replication forks, and synthesis is bidirectional, so bubbles expand in both directions.

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.

Synthesis of the Leading Strand

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 initiate synthesis de novo; it requires a free 3' end provided by a short RNA primer.

• Primase (an RNA polymerase) synthesizes the primer.

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

Synthesis of the Lagging Strand

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 and their functions:

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 and prevent strands from rejoining.
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.

The Replisome

The replisome is a large macromolecular complex containing all the enzymes and proteins required for DNA synthesis at the replication fork. It is dynamic, and recent observations suggest that the synthesis of the continuous strand may not be as continuous as previously thought, with rates of DNA synthesis varying widely.

Replicating the Ends of Linear Chromosomes

The End Replication Problem and Telomeres

Replication of the ends of linear chromosomes (telomeres) presents a unique challenge:

• The leading strand can be synthesized to the end, but the lagging strand cannot be fully replicated because there is no primer for DNA polymerase to extend.

• This leaves a single-stranded overhang, which is eventually degraded, causing chromosomes to shorten with each cell division.

• Telomeres are repetitive, non-coding sequences that protect chromosome ends from loss of genetic information.

Telomerase and Telomere Maintenance

Telomerase is an enzyme that extends telomeres by adding repetitive DNA sequences using an RNA template it carries:

• Telomerase binds to the overhang and extends it, allowing normal DNA synthesis to complete the lagging strand.

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

• In the absence of telomerase, chromosomes shorten with each division, limiting the number of cell divisions (the Hayflick limit).

• Most cancer cells reactivate telomerase, enabling unlimited division.

Correcting Mistakes in DNA Synthesis

DNA Polymerase Proofreading

DNA polymerase has proofreading ability, ensuring high fidelity during DNA replication:

• Incorrectly paired bases are detected due to their distinct shape.

• The enzyme’s exonuclease site removes mismatched nucleotides, allowing correct bases to be inserted.

Mismatch Repair

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

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

Repairing Damaged DNA

DNA can be damaged by environmental factors such as UV light, X-rays, and chemicals. Organisms have evolved repair systems to maintain genome integrity:

• 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 section, and uses the intact strand as a template for repair. DNA ligase seals the repaired strand.

Summary Table: DNA Repair Mechanisms

Mechanism	When It Occurs	Key Enzymes	Function
Proofreading	During DNA synthesis	DNA polymerase	Removes mismatched nucleotides immediately
Mismatch repair	After DNA synthesis	Mismatch repair enzymes	Removes and replaces incorrect bases
Nucleotide excision repair	Anytime DNA is damaged	Excision repair complex, DNA ligase	Removes and replaces damaged DNA sections

Additional info: The accuracy of DNA replication and repair is essential for genetic stability. Defects in these systems can lead to mutations, cancer, and genetic diseases.