13.5: Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell’s circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from two replication forks, resulting in two DNA molecules.

Many Proteins Work Together to Replicate the Chromosome

Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands. Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. DNA can only be synthesized in the 5’ to 3’ direction, so one strand of the template—the leading strand—is elongated continuously, while the other strand—the lagging strand—is synthesized in shorter pieces of 1000-2000 base pairs called Okazaki fragments.

Multiple Polymerases Take Part in Elongation

Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli, a commonly-used model organism. E. coli has 5 DNA polymerases: Pol I, II, III, IV, and V. Pol III is responsible for the majority of DNA replication. It can polymerize about 1,000 base pairs per second. This astonishing pace allows the machinery present at the two replication forks to duplicate the E. coli chromosome—4.6 million base pairs—in roughly 40 minutes. DNA polymerase I is also well-characterized; its primary role is to remove the RNA primers from the start of Okazaki fragments on the lagging strand.

When Division Outpaces Duplication

Under favorable growth conditions, E. coli will divide every 20 minutes, about half the amount of time that it takes to replicate the genome. How is this possible when both daughter cells must have their own DNA? Scientists found that the bacteria can begin another round of DNA replication from the origin of replication before the first round is complete; this means that daughter cells receive a chromosome that is already in the process of being copied and are prepared to divide again very quickly.

In prokaryotes, DNA replication begins when initiator proteins bind to the origin of replication, a small region of DNA containing a specific sequence of bases, creating a complex.

This complex helps to initially separate the DNA. Then the enzyme DNA helicase binds to it and continues to unwind the DNA by breaking the hydrogen bonds between the complementary strands. The newly opened areas are stabilized by single-stranded DNA binding proteins. Each one can now serve as a template for the synthesis of a new strand of DNA.

The unwinding and synthesis proceeds in both directions from the origin, creating two replication forks. In front of the forks, topoisomerase enzymes bind to the DNA and reduce torsional strain as the molecule unwinds.

Once the strands are separated, another enzyme, primase, synthesizes an RNA primer, a short stretch of RNA complementary to the DNA sequence. The primer provides a place for the enzyme DNA polymerase to add nucleotides complementary to the DNA sequence, creating a new DNA strand in a process called elongation.

DNA polymerase synthesizes DNA in the five prime to three prime direction of the molecule, so the synthesis of this strand, the leading strand, proceeds continuously. The other strand, the lagging strand, has the opposite orientation. Consequently DNA is synthesized in short pieces called Okazaki fragments, elongated from additional RNA primers backwards from the overall direction of movement of the replication fork.

The RNA primers are then excised by enzymes such as RNAs, replaced with DNA, and the DNA fragments are joined together by the enzyme DNA ligase, creating a continuous strand.

DNA replication proceeds around the entire molecule, resulting in two circular DNA molecules. This is considered a semiconservative process, because each molecule contains one old strand and one new strand.