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 strand. Each daughter DNA molecule consists of one old (parental) 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 parental DNA is cut into 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 synthesis is catalyzed by enzymes called DNA polymerases. Several types exist, but all share key properties:

• DNA polymerases can only add nucleotides to the 3' end of a growing DNA strand, so synthesis always proceeds in the 5' to 3' direction.

• The building blocks are deoxyribonucleoside triphosphates (dNTPs), which have high potential energy due to their three phosphate groups. Hydrolysis of these phosphates makes the formation of phosphodiester bonds exergonic.

Equation: The general reaction for DNA polymerization is: $\text{(dNMP)}_n + \text{dNTP} \rightarrow \text{(dNMP)}_{n+1} + \text{PP}_i$

Where Does Replication Start?

DNA replication begins at specific locations called origins of replication:

• In bacteria, there is 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 synthesis proceeds bidirectionally.

How is the Helix Opened and Stabilized?

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 caused by unwinding by cutting and rejoining DNA ahead of the replication fork.

How Is the Leading Strand Synthesized?

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 strand in the 5' to 3' direction.

How is the Lagging Strand Synthesized?

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

Multiple proteins coordinate DNA replication. The following table summarizes their names and 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
Topoisomerase	Relieves twisting forces caused by the opening of the helix
Primase	Catalyzes the synthesis of the 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 research suggests that the process is more flexible and variable than previously thought.

Replicating the Ends of Linear Chromosomes

Replication of telomeres (the ends of eukaryotic chromosomes) 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 the final segment.

• This results in progressive shortening of chromosomes with each cell division.

• Telomeres consist of short, repeating, non-coding DNA sequences.

Telomerase and the End Replication Problem

Telomerase is an enzyme that extends the ends of chromosomes, solving the end replication problem:

• It carries its own RNA template and adds DNA repeats to the 3' end of the lagging strand template.

• Once the overhang is long enough, normal DNA polymerase can complete replication.

• 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 is highly accurate, but mistakes can occur. Several mechanisms ensure fidelity:

• Proofreading: DNA polymerase has an exonuclease activity that removes mismatched nucleotides immediately after they are added.

• Mismatch repair: Specialized enzymes recognize and repair mismatches left after replication is complete.

Repairing Damaged DNA

DNA can be damaged by environmental factors such as UV light and chemicals. Organisms have evolved repair systems:

• Nucleotide excision repair: Recognizes distortions in the DNA helix (e.g., thymine dimers), removes the damaged section, and fills in the gap using the undamaged strand as a template. DNA ligase seals the repaired strand.

Summary Table: DNA Repair Mechanisms

Mechanism	When It Acts	Key Enzymes
Proofreading	During DNA synthesis	DNA polymerase (exonuclease activity)
Mismatch repair	Immediately after DNA synthesis	Mismatch repair enzymes
Nucleotide excision repair	Anytime DNA is damaged	Excision repair complex, DNA polymerase, DNA ligase

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