All organisms reproduce, it is one of the main features of life. If an organism is just a cell, it can reproduce very quickly. For example, bacteria can double every 20 minutes! But before a bacterial cell divides into two new ones, it has to solve an important problem. Every cell needs its own DNA, so where can it get enough DNA to make two daughter cells? The cells of our body are much more complex than bacteria and reproduce slower, but they have to solve the same problem. However, for both of them, the answer will be the same. Replication. Let's see what it's all about.
Replication
DNA replication is a process that facilitates the copying of a double-stranded DNA molecule. The result is two new DNA molecules that ideally are exact copies of the original molecule. Though, these two molecules are not entirely new. As you remember from the Nucleic acids topic, DNA is a double-stranded structure. After replication, each unique DNA molecule consists of a strand from the original DNA molecule and a newly synthesized strand. This is because, during replication, each strand of the original molecule serves as a template for DNA polymerase, which does the synthesis of its complementary strand. In short, replication is semiconservative.
DNA polymerase
The new strand of DNA is synthesized by an enzyme called DNA polymerase. It moves along the already existing DNA strand and adds complementary nucleotides to the new strand one by one. DNA polymerase has several essential features:
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It needs a template.
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It needs a primer, a short stretch of nucleotides because DNA polymerase only can add a nucleotide to another nucleotide.
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It can add a nucleotide to the 3' end of the other. Since strands in the DNA molecule are antiparallel, it means that DNA polymerase moves from the 5' end to the 3' end of the template.
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It has a proofreading activity. If DNA polymerase makes a mistake and adds the wrong not-complementary nucleotide to a new stand, it can remove it and fix the strand. In this way DNA polymerase prevents mutations.
Like any chemical synthesis, replication requires energy. It comes from nucleotides themselves. When nucleotides enter the replication process, they bear three attached phosphates. DNA polymerase breaks off the two extreme phosphates, and the energy released as a result of breaking the bond is spent on creating a new bond between the remaining phosphate and the -OH group of the sugar of the previous nucleotide. The principle is the same as in the case of the ATP energy molecule (see Cell structure topic).
Initiation: start of DNA replication
DNA replication starts at particular points on the DNA strand, which are known as the origins of replication. The region of DNA which is copied from a single origin is called a replication unit or a replicon. In the case of a bacterial cell, its whole chromosome is a replicon.
Replication enzymes recognize origins by a sequence that usually is rich with A/T base pairs. Since the A/T base pair has only two hydrogen bonds (the G/C has three), the connection between DNA strands in the origin is not very strong.
When special initiator proteins recognize an origin and bind to it, they recruit more proteins to form a replication complex. One of these proteins is a helicase, which unwinds the DNA helix breaking hydrogen bonds between base pairs. Thus, the DNA "opens" into a replication bubble made of two replication forks. As the replication progresses, the forks will move away from each other. To prevent unwinded and separated DNA strands from joining again into the double helix, special proteins called single-strand binding proteins cover them near the point of the fork.
Separated DNA strands now can serve as templates for replication, but before DNA polymerase starts working, another special enzyme called primase makes an RNA primer. Primers are short (5-10 nucleotides) and complementary to a template. They provide a DNA polymerase with a 3' end to work on. Once the primers are ready, the initiation step of the replication is over. DNA polymerase is able to attach the template and extend a primer into a new strand. The elongation step of the replication starts.
Elongation: leading and lagging strands
A replication fork consists of two strands, and each strand needs its own DNA polymerase III. Unfortunately, one of the polymerases faces a problem. As you have seen earlier, DNA polymerase can only add nucleotides to the 3' end and move to the 5' end of the template (remember, strands in the DNA are antiparallel). It is easy to do when polymerase makes a so-called leading strand, which has its 3' end directed towards the fork. The fork moves constantly, and so does DNA polymerase, moving in the same direction and synthesizing a new strand continuously.
However, another strand runs away from the fork, and the fork and this DNA polymerase move in opposite directions. That's why DNA polymerase of a lagging strand should constantly fall off and reattach when another part of the template is exposed. Thus, the lagging strand is made of short fragments called Okazaki fragments, named for a Japanese scientist who discovered them. To start the synthesis of an Okazaki fragment, DNA polymerase needs a new primer. In contrast, the leading strand needs just one primer.
Several proteins help DNA polymerase to work. One is the protein called the sliding clamp, which holds DNA polymerase on the template. It also doesn't let polymerase float far away when it falls off to start a new Okazaki fragment.
Another protein is a topoisomerase, which prevents the DNA ahead of the replication fork from super-coiling. To release the tension it makes temporary nicks in the DNA helix and heals them after.
There also should be someone to clean the mess. DNA polymerase I removes RNA primers and synthesizes a DNA sequence instead. Then, remained nicks in DNA strands are healed by DNA ligase.
Termination: end of DNA replication
Elongation stops when replication forks meet. A bacterial cell regulates the termination of replication by a special termination sequence, which recruits proteins blocking DNA polymerase movement. That's why replication forks always meet within the termination sequence in the case of bacteria. Since the bacterial chromosome is circular, termination occurs on the opposite of the original sequence.
Conclusion
DNA replication is a process of producing two copies of a DNA molecule. Each copy consists of a strand from the original molecule and a strand synthesized anew. Initiation of replication occurs in the origin sites where DNA strands unwind and separate. It leads to the formation of a replication fork consisting of two DNA strands, which serve as templates for replication. An enzyme called DNA polymerase synthesizes new DNA sequences adding nucleotides to the 3' end of the growing strand. DNA elongation starts from the RNA primer. Elongation is continuous in the leading strand, but the lagging strand is made of short fragments. Once elongation is complete, RNA primers are removed, nicks are closed, and replication terminates.