DNA holds the genetic instructions that dictate the development and function of all living organisms. For a cell to grow, divide, or repair damaged tissues, its DNA must be accurately copied. This process, known as DNA replication, ensures that each new cell receives a complete and identical set of genetic information from the parent cell.
Locating the Start
DNA replication starts at specific sites called “origins of replication” (ORIs). These ORIs are particular DNA sequences recognized by specialized proteins. In bacteria, a single origin typically exists on their circular chromosome, while eukaryotes have multiple origins for efficient duplication.
Initiator proteins recognize and bind to these origins. For instance, in E. coli, the DnaA protein binds to specific sequences within the oriC region, which often contains a higher proportion of adenine (A) and thymine (T) base pairs. A-T rich regions are easier to separate because they are held together by two hydrogen bonds, compared to the three hydrogen bonds between guanine (G) and cytosine (C) base pairs. This binding causes a small section of the DNA double helix to unwind and separate, forming a “replication bubble.” Each replication bubble contains two “replication forks” that move in opposite directions, allowing for bidirectional copying of the DNA.
Unwinding the Double Helix
Following the initial recognition and localized unwinding, the next step involves the physical separation of the DNA strands. This unwinding is primarily carried out by an enzyme called DNA helicase. DNA helicase moves along the DNA, effectively “unzipping” the double helix by breaking the hydrogen bonds that connect the complementary base pairs. This action requires energy, which DNA helicase obtains from the hydrolysis of ATP.
As DNA helicase unwinds the double helix, it creates a Y-shaped structure known as a replication fork. The continuous unwinding of the DNA ahead of the replication fork introduces torsional stress, causing the DNA molecule to become overwound or “supercoiled.” If this stress is not alleviated, it would impede the progression of the replication fork and halt the replication process.
To counteract this supercoiling, another class of enzymes called DNA topoisomerases. These enzymes relieve the torsional strain by temporarily cutting the DNA strands and then rejoining them. In bacteria, a specific type of topoisomerase known as DNA gyrase actively introduces negative supercoils and relaxes positive supercoils that accumulate ahead of the replication fork. This coordinated action of helicase and topoisomerase ensures the DNA remains accessible for subsequent replication steps.
Keeping Strands Separate
Once the DNA double helix is unwound, the separated single strands are unstable and tend to re-anneal, or re-form hydrogen bonds. To prevent this, single-strand binding proteins (SSBs) quickly attach to the exposed single strands of DNA.
SSBs bind to the single-stranded DNA in a sequence-independent manner, effectively stabilizing them. Their presence prevents the separated strands from coming back together, which would otherwise block the replication machinery. SSBs also protect the vulnerable single-stranded DNA from degradation by enzymes. By keeping the strands separated and stable, single-strand binding proteins ensure that each single strand can serve as a proper template for the synthesis of new DNA.