DNA Replication: Mechanisms and Enzymes

DNA Replication

Overview of DNA Replication

DNA replication is the biological process by which a cell duplicates its DNA, ensuring that each daughter cell receives an exact copy of the genetic material. This process is essential for cell division and inheritance. Replication is semiconservative, meaning each new DNA molecule consists of one parental and one newly synthesized strand.

• Template strands: Parental DNA strands serve as templates for the synthesis of new complementary strands.

• Separation of strands: The two DNA strands must be separated to allow copying; this is achieved by breaking hydrogen bonds between base pairs.

• Replication origins: Replication begins at specific sites called origins of replication.

Initiation of Replication

Replication starts at origins of replication, where specialized proteins recognize specific DNA sequences and recruit helicase enzymes to unwind the DNA.

• Replication bubble: The region where the DNA is unwound forms a replication bubble with two replication forks moving in opposite directions.

• Helicase: An enzyme that unwinds the DNA double helix by breaking hydrogen bonds between base pairs, creating single-stranded regions for copying.

• AT-rich regions: Origins often contain more A-T base pairs, which are easier to separate due to having only two hydrogen bonds (compared to three in G-C pairs).

Prokaryotic vs. Eukaryotic Replication Origins

• Prokaryotes: Typically have a single origin of replication per circular chromosome.

• Eukaryotes: Possess multiple origins per linear chromosome to ensure timely replication of large genomes.

Maintaining Structural Integrity During Polymerization

Unwinding DNA creates single-stranded regions that are prone to re-annealing or degradation. Cells use specific proteins to stabilize these regions and prevent unwanted interactions.

• Single-stranded binding proteins (SSBs): Bind to separated DNA strands, preventing them from re-annealing or forming secondary structures.

• Function: SSBs create a physical barrier, protecting single-stranded DNA and allowing replication enzymes to function efficiently.

Relieving Torsional Strain: Topoisomerases

As helicase unwinds DNA, it introduces supercoiling (overwinding) ahead of the replication fork, which can impede replication. Topoisomerases are enzymes that relieve this strain by cutting and rejoining DNA strands.

• Topoisomerase I: Introduces single-strand breaks, allowing the DNA to rotate and relieve supercoiling. It acts at one location and does not require ATP.

• Topoisomerase II (e.g., DNA gyrase in prokaryotes): Introduces double-strand breaks, passes another segment of the DNA through the break, and reseals it. This process requires ATP and is essential for managing higher levels of supercoiling.

Polymerization: Assembling the Daughter Strands

New DNA strands are synthesized by DNA polymerases, which add nucleotides to a growing DNA chain using the parental strand as a template. This process is called polymerization.

• Directionality: DNA polymerases synthesize new DNA in the 5' to 3' direction only.

• Energy source: The energy for polymerization comes from the hydrolysis of the high-energy phosphate bonds in deoxynucleoside triphosphates (dNTPs).

• Equation:

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

• Primase: Synthesizes short RNA primers that provide a free 3'-OH group for DNA polymerase to begin synthesis.

• Requirement for primers: DNA polymerases cannot initiate synthesis de novo; they require an existing primer.

Leading and Lagging Strand Synthesis

Because DNA polymerase can only add nucleotides in the 5' to 3' direction, the two new strands are synthesized differently:

• Leading strand: Synthesized continuously in the same direction as the replication fork movement.

• Lagging strand: Synthesized discontinuously in short fragments called Okazaki fragments, which are later joined by DNA ligase.

Process of Lagging Strand Synthesis

• Primase synthesizes multiple RNA primers along the lagging strand template.

• DNA polymerase extends each primer, creating Okazaki fragments.

• RNA primers are removed and replaced with DNA.

• DNA ligase seals the nicks between fragments, forming a continuous strand.

Enzymes Involved in DNA Replication

• Helicase: Unwinds the DNA double helix.

• Single-stranded binding proteins (SSBs): Stabilize unwound DNA.

• Primase: Synthesizes RNA primers.

• DNA polymerase: Synthesizes new DNA strands; different types exist in prokaryotes and eukaryotes.

• Topoisomerase: Relieves supercoiling ahead of the replication fork.

• DNA ligase: Joins Okazaki fragments on the lagging strand.

Types of DNA Polymerases

• Prokaryotes: Main DNA polymerases are DNA pol I, II, and III (with pol III being the primary replicative enzyme).

• Eukaryotes: Multiple DNA polymerases, labeled with Greek letters (e.g., α, δ, ε), each with specialized functions.

Summary Table: Key Enzymes and Their Functions

Enzyme	Function
Helicase	Unwinds the DNA double helix at the replication fork
Single-stranded binding protein (SSB)	Stabilizes single-stranded DNA and prevents re-annealing
Primase	Synthesizes short RNA primers for DNA polymerase
DNA polymerase	Adds nucleotides to the growing DNA strand
Topoisomerase I	Relieves supercoiling by introducing single-strand breaks
Topoisomerase II (DNA gyrase)	Relieves supercoiling by introducing double-strand breaks
DNA ligase	Joins Okazaki fragments on the lagging strand

Key Concepts and Definitions

• Origin of replication: Specific DNA sequence where replication begins.

• Replication fork: The Y-shaped region where the DNA is split into two single strands for copying.

• Okazaki fragments: Short DNA fragments synthesized on the lagging strand.

• Semiconservative replication: Each new DNA molecule contains one parental and one new strand.

• Antiparallel: The two strands of DNA run in opposite directions (5' to 3' and 3' to 5').

Example: DNA Replication in E. coli

• Replication begins at a single origin (oriC) and proceeds bidirectionally around the circular chromosome.

• DNA polymerase III synthesizes most of the new DNA, while DNA polymerase I removes RNA primers and fills in the gaps.

• DNA ligase seals the nicks to complete the process.

Additional info: Eukaryotic chromosomes are linear and much larger than prokaryotic chromosomes, necessitating multiple origins of replication to ensure the entire genome is copied efficiently during S phase of the cell cycle.