3-4-5. DNA Replication in Prokaryotes
DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistakes.
DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.
How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called
You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?
The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5' to 3' direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction. One strand, which is complementary to the 3' to 5' parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the
The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows:
Table 1. Prokaryotic DNA Replication: Enzymes and Their Function
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Review the full process of DNA replication here.
Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication fork; this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand.
Figure 1. You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?
Figure 1. DNA ligase, as this enzyme joins together Okazaki fragments.
Which of the following components is not involved during the formation of the replication fork?
Which of the following does the enzyme primase synthesize?
In which direction does DNA replication take place?
DNA replication is bidirectional and discontinuous; explain your understanding of those concepts.
At an origin of replication, two replication forks are formed that are extended in two directions. On the lagging strand, Okazaki fragments are formed in a discontinuous manner.
What are Okazaki fragments and how they are formed?
Short DNA fragments are formed on the lagging strand synthesized in a direction away from the replication fork. These are synthesized by DNA pol.
If the rate of replication in a particular prokaryote is 900 nucleotides per second, how long would it take 1.2 million base pair genomes to make two copies?
1333 seconds or 22.2 minutes.
Explain the events taking place at the replication fork. If the gene for helicase is mutated, what part of replication will be affected?
At the replication fork, the events taking place are helicase action, binding of single-strand binding proteins, primer synthesis, and synthesis of new strands. If there is a mutated helicase gene, the replication fork will not be extended.
What is the role of a primer in DNA replication? What would happen if you forgot to add a primer in a tube containing the reaction mix for a DNA sequencing reaction?
Primer provides a 3'-OH group for DNA pol to start adding nucleotides. There would be no reaction in the tube without a primer, and no bands would be visible on the electrophoresis.
single-strand binding protein