The replication of dsDNA involves the separation of the two strands, allowing each strand to act as a template for the synthesis of a new complementary strand. This process results in the production of two daughter molecules, each containing two DNA strands (one old and one new) in an antiparallel orientation. It is known as semiconservative replication because although the parental duplex is split into two halves, each parental strand remains intact in one of the new duplexes. The enzymes involved in DNA replication are template-directed, magnesium-requiring polymerases that have a high level of accuracy in synthesizing the complementary sequence of each strand. Initially studied in Escherichia coli (E. coli), the replication process in prokaryotes is described here. However, DNA synthesis in higher organisms follows similar mechanisms, although it is more complex. Once DNA replication is initiated, the cell is committed to replicating the entire genome.

A. Complementary strand separation:
To replicate the two complementary strands of parental dsDNA, they must first separate over a small region in a process known as "melt." This is necessary because the polymerases only use single-stranded DNA (ssDNA) as a template. In prokaryotic organisms, DNA replication starts at a single nucleotide sequence called the origin of replication or ori (oriC in E. coli). This sequence is referred to as a consensus sequence as it has a similar order of nucleotides at each site. The ori contains short, AT-rich segments that facilitate the separation of the strands. In eukaryotes, replication initiates at multiple sites along the DNA helix, allowing for the efficient replication of the lengthy eukaryotic DNA molecules.

B. Replication fork formation:
As the two strands unwind and separate, replication occurs at two replication forks that move bidirectionally away from the origin, leading to the formation of a replication bubble. The term "replication fork" derives from the Y-shaped structure formed by the separated strands. Initiation of DNA replication requires a group of proteins that form the prepriming complex and recognize the origin as the start site. These proteins are responsible for melting at the ori, maintaining strand separation, and unwinding the double helix ahead of the replication fork. In E. coli, these proteins include DnaA protein, which binds to specific nucleotide sequences (DnaA boxes) within oriC and causes melting of an AT-rich region known as the DNA unwinding element. This results in the formation of a short, localized region of single-stranded DNA. DNA helicases, another group of proteins, bind to the single-stranded DNA near the replication fork and move into the adjacent double-stranded region, forcing the strands apart and unwinding the double helix. ATP hydrolysis provides the energy required for helicase activity. Unwinding at the replication fork leads to supercoiling in other regions of the DNA molecule. In E. coli, the principal helicase of replication is DnaB, which requires DnaC for binding to DNA.
When the two strands of dsDNA are separated, each can serve as a template for the replication (synthesis) of a new complementary strand. This produces two daughter molecules, each of which contains two DNA strands (one old, one new) in an antiparallel orientation (see Fig. 30.3). This process is called semiconservative replication because, although the parental duplex is separated into two halves (and, therefore, is not conserved as an entity), each of the parental strands remains intact in one of the two new duplexes (Fig. 30.8). The enzymes involved in DNA replication are template-directed, magnesium (Mg2+)requiring polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. The reactions described in this section were first known from studies of the bacterium Escherichia coli (E. coli), and the description given below refers to the process in prokaryotes. DNA synthesis in higher organisms is more complex but involves the same types of mechanisms. In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated.
A. Complementary strand separation
In order for the two complementary strands of the parental dsDNA to be replicated, they must first separate (or “melt”) over a small region, because the polymerases use only ssDNA as a template. In prokaryotic organisms, DNA replication begins at a single, unique nucleotide sequence, a site called the origin of replication, or ori (oriC in E. coli), as shown in Figure 30.9A. [Note: This sequence is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.] The ori includes short, AT-rich segments that facilitate melting. In eukaryotes, replication begins at multiple sites along the DNA helix (Fig. 30.9B). Having multiple origins of replication provides a mechanism for rapidly replicating the great length of eukaryotic DNA molecules.
B. Replication fork formation
As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally), generating a replication bubble (see Fig. 30.9). [Note: The term “replication fork” derives from the Y-shaped structure in which the tines of the fork represent the separated strands (Fig. 30.10).] 1. Required proteins: Initiation of DNA replication requires the recognition of the origin (start site) by a group of proteins that form the prepriming complex. These proteins are responsible for melting at the ori, maintaining the separation of the parental strands, and unwinding the double helix ahead of the advancing replication fork. In E. coli, these proteins include the following.
a.
DnaA protein: DnaA protein initiates replication by binding to specific nucleotide sequences (DnaA boxes) within oriC. Binding causes an AT-rich region (the DNA unwinding element) in the origin to melt. Melting (strand separation) results in a short, localized region of ssDNA.
b.
DNA helicases: These enzymes bind to ssDNA near the replication fork and then move into the neighboring double-stranded region, forcing the strands apart (in effect, unwinding the double helix). Helicases require energy provided by ATP hydrolysis (see Fig. 30.10). Unwinding at the replication fork causes supercoiling in other regions of the DNA molecule. [Note: DnaB is the principal helicase of replication in E. coli. Binding of this hexameric protein to DNA requires DnaC.] c.