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 hypotheses 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 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 chain, 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 groups makes the formation of phosphodiester bonds exergonic.

Equation:

$\mathrm{(dNMP)_n + dNTP \rightarrow (dNMP)_{n+1} + PP_i}$

Additional info: The energy released from breaking the phosphate bonds in dNTPs drives the polymerization reaction.

Where Does Replication Start?

DNA replication begins at specific locations called origins of replication:

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

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

• Each bubble contains two replication forks that move in opposite directions, making replication bidirectional.

How is the Helix Opened and Stabilized?

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

• 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 downstream 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 initiate synthesis de novo; it requires a free 3' hydroxyl group.

• A short RNA primer is synthesized by primase (an RNA polymerase) and provides the starting point.

• 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 the process of DNA replication. The following table summarizes their names and functions:

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 it from re-forming a double helix.
Topoisomerase	Relieves twisting forces caused by the opening of the helix.
Primase	Synthesizes RNA primer for DNA polymerase.
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: DNA-Synthesizing Machine

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 synthesis of the continuous strand may not be as continuous as previously thought, with variable rates of DNA synthesis.

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 single-stranded DNA overhangs, which are eventually degraded, causing chromosomes to shorten with each cell division.

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

Telomerase and the End Replication Problem

Telomerase is an enzyme that extends telomeres using an RNA template it carries:

• It binds to the overhang and adds repeating DNA sequences, allowing normal DNA polymerase to complete the lagging strand.

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

• In somatic cells, telomere shortening limits the number of cell divisions, which is thought to contribute to aging.

• Most cancer cells reactivate telomerase, enabling unlimited division.

Correcting Mistakes in DNA Synthesis

DNA polymerase is highly accurate, but mistakes can occur:

• Correct base pairs are energetically favored and have a distinct shape.

• DNA polymerase inserts an incorrect base about once every 100,000 bases, but repair enzymes correct most errors.

DNA Polymerase Proofreading

DNA polymerase has proofreading ability:

• Mismatched bases are detected by their abnormal shape.

• The enzyme’s exonuclease site removes the incorrect nucleotide, allowing synthesis to continue with the correct base.

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:

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

• The nucleotide excision repair system recognizes and removes damaged DNA, using the undamaged strand as a template for repair. DNA ligase then seals the repaired strand.