Every time a cell divides, each new daughter cell must be supplied with a copy of the cell’s genetic material, meaning that the genome of a cell must be accurately duplicated—a process known as replication—each generation. While the specifics we discuss refer to replication in prokaryotic organisms, the general process is similar in eukaryotic organisms, and to a lesser extent, in viruses.
The Overall Replication Process
What features allow the DNA molecule to be exactly duplicated, and how is its integrity retained? DNA replication requires a careful orchestration of the actions of 30 different enzymes (see the partial list in table 1), which separate the strands of the existing DNA molecule, copy each strand, and produce two complete daughter molecules.
Table1. Some Enzymes Involved in DNA Replication and Their Functions
A simplified version of replication is shown in figure 1 and includes the following:
1. uncoiling the parent DNA molecule, beginning at a predetermined point of origin;
2. separating the two strands and exposing the nucleotide sequence of each strand (which is normally hidden in the center of the helix) to serve as templates; and
3. synthesizing two new strands by using each single strand as a template for the synthesis of a new, complementary strand.
Fig1. Replication of DNA relies on each strand of the molecule serving as a template for synthesis of a new strand. Each single strand will serve as a template (blue) to synthesize a new strand of DNA (red), starting at the replication fork. As synthesis proceeds, the correct nucleotides are added according to the pattern of the template. An A on the template will pair with a T on the new molecule, and a C will pair with a G.
A critical requirement of DNA replication is that each completed daughter molecule be identical to the parent in composition, but neither one is completely new. The strand that serves as a template is an original parental DNA strand and is retained in the daughter molecule. The preservation of the parent molecule in this way, termed semiconservative replication, helps explain the reliability and accuracy of replication (figure 2).
Fig2. Replication of the bacterial chromosome.
Events in Replication
All chromosomes have a specific origin of replication that serves as the place where replication will be initiated. It is recognized by a short sequence rich in adenine and thymine that, you will recall, are held together by only two hydrogen bonds rather than three. Because the origin of replication is AT-rich, less energy is required to separate the two strands than would be required if the origin were rich in guanine and cytosine.
The process of synthesizing a new daughter strand of DNA using the parental strand as a template is carried out by a giant enzyme complex that brings in the primary replication enzyme—DNA polymerase III—along with numerous accessory enzymes. DNA polymerase III is responsible for duplicating DNA, but it has some major conditions that will affect the overall process (figure 3):
● DNA polymerase can only add nucleotides to an existing nucleotide. It is unable to begin the process on its own. The enzyme primase will synthesize a short stretch of nucleotides, allowing DNA polymerase to begin its work.
● Nucleotides may only be added to a growing DNA chain in the 5′ to 3′ direction.
Fig3. DNA polymerase III The DNA polymerase III molecule is a huge complex composed of more than a dozen individual proteins, which together work to replicate the DNA molecule. Part of the enzyme serves as a DNA clamp, keeping the polymerase attached to the DNA as replication proceeds. Other portions of the enzyme check for errors in the newly made DNA (proofreading) and replace incorrect nucleotides when they are found. McGraw-Hill Education
Prior to the start of replication, enzymes called helicases bind to the DNA at the origin. These enzymes untwist the helix and break the hydrogen bonds holding the two strands together, resulting in two separate strands, each of which will be used as a template for the synthesis of a new strand (process figure 4, step 1).
Fig4. Replication of a circular bacterial chromosome. An overall view of the chromosome is seen at top, with detail of the replication bubble below. Barry Chess/McGraw Hill
Replication begins when RNA primers are synthesized by a primase at the origin of replication (process figure 4). DNA polymerase III will use this short strand of RNA as a starting point for adding nucleotides. In the circular DNA molecule of bacteria, there are two replication forks, each containing its own set of replication enzymes. Each fork requires two active DNA polymerases along with several other proteins and enzymes whose main functions are to stabilize the polymerase and provide a means of removing and replacing nucleotides. The enzyme complex encircles the DNA near the replication fork and synthesizes new DNA as guided by the parental strand. As synthesis proceeds, the forks are continually opened up to expose the template for replication.
Because DNA polymerase III can only synthesize new DNA in the 5′ to 3′ direction, just one strand, called the leading strand, will be synthesized continuously (process figure 4, step 2). After addition of an RNA primer, a DNA polymerase complex enters and begins to synthesize a new DNA strand in the 5’ to 3’ direction (note that this means that the template DNA is being read in the 3’ to 5’ direction).
The other new strand, termed the lagging strand, will be synthesized in a series of short fragments that will later be connected into a continuous strand (process figure4, steps 3 and 4). It still requires primers, and each new fragment will require its own separate primer. The DNA polymerase adds nucleotides to the new strand in the 5′ to 3′ direction, producing short fragments of DNA (100 to 1,000 bases long) called Okazaki fragments, which together make up the lagging strand. DNA polymerase I then re moves the RNA nucleotides that make up the primer and replaces them with corresponding DNA nucleotides. The enzyme ligase acts next, catalyzing a sugar-phosphate bond between the Okazaki fragments, chemically bonding them to one another and producing a fully replicated DNA molecule (process figure 4, step 5).
The addition of nucleotides proceeds at an astonishing pace, estimated in some bacteria to be 750 bases per second at each fork! As replication proceeds, one newly synthesized strand loops down (figure 2). When the forks have gone full circle, a termination site shuts replication down. The two circular daughter molecules remain connected briefly but are nicked and separated by enzymes within the cell.
The double-stranded nature of DNA and the rules of base pairing (adenine to thymine and guanine to cytosine) ensure that the correct order of the DNA bases will be retained during cell division. When the two strands are separated, each one provides a template (pattern or model) for the replication (exact copying) of a new molecule (figure1). Because the sequence of one strand provides the correct pattern for its complementary strand, the code can be duplicated with accuracy.
Like any language, DNA is occasionally “misspelled” when an incorrect base is added to the growing chain. Studies have shown that such mistakes are made once in approximately 108 to 109 bases, but most of these are corrected. If not corrected, they are considered mutations that can lead to major cell dysfunction. Because continued cellular function is dependent on accurate replication, cells have evolved their own proofreading ability. Both DNA polymerase III and DNA polymerase I have the ability to recognize and replace incorrect nucleotides in newly synthesized DNA.
