DNA Replication
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During cell division, each daughter cell requires a copy of the genetic information from the parental cell, necessitating DNA replication prior to division. The fundamental principles of DNA replication are dictated by the structure of DNA, which comprises two complementary strands. According to the semi-conservative replication model, each strand of the DNA serves as a template for the synthesis of a new strand. As a result, after replication, each daughter DNA molecule contains one original (old) strand and one newly synthesised strand.
While the major principles of DNA replication are similar in eukaryotes and prokaryotes, this chapter will focus on the process as it occurs in bacteria, such as Escherichia coli, due to its relative simplicity.
DNA synthesis initiates at specific sites known as replication origins. Most bacteria possess a single circular chromosome with only one replication origin. Upon the separation of DNA strands at the origin, a replication bubble forms. Replication starts simultaneously in two directions from the origin, leading to the formation of two replication forks. These forks move away from each other, performing DNA synthesis in their respective directions.
At the replication fork, DNA synthesis is catalyzed by enzymes known as DNA polymerases. These polymerases require a single-stranded DNA template, which remains in the newly synthesized DNA as the original DNA strand. The substrates for the DNA polymerase reaction include the elongating new DNA strand and free nucleotides in the form of deoxyribonucleoside triphosphates. These nucleotides serve as the building blocks for the growing DNA strand, with each nucleotide being incorporated into the elongating DNA chain in a template-directed manner.
At the replication fork, the two template DNA strands are anti-parallel, with one running in the 5'-to-3' direction and the other in the 3'-to-5' direction. However, DNA polymerases can only elongate new DNA strands in the 5'-to-3' direction. Consequently, one DNA strand, known as the leading strand, can be synthesised continuously towards the replication fork.
The other strand, termed the lagging strand, must be synthesised in a discontinuous manner. DNA polymerase moves away from the replication fork, reading the template, and synthesises short fragments called Okazaki fragments. After each fragment is synthesised, the DNA polymerase detaches from the template and attaches to a new segment of single-stranded DNA cleared by the progressing replication fork. In bacteria, Okazaki fragments are typically 1000-2000 nucleotides long, whereas in eukaryotes, they are approximately 10 times shorter.
In addition to DNA polymerase, DNA replication involves numerous other proteins. The movement of the replication fork is facilitated by helicase, which unwinds the double helix, separating the template strands from each other.
Immediately following unwinding, the template strands are bound by single-strand DNA binding (SSB) proteins, preventing them from reannealing. This ensures the accessibility of the template strands for replication.
The unwinding of the double helix by helicase generates supercoiling, excessive twisting, in front of the replication fork. This supercoiling is relieved by another enzyme called topoisomerase.
DNA replication initiation poses a challenge as DNA polymerase cannot initiate the synthesis of a new polynucleotide; it can only elongate an existing one. To overcome this, another enzyme, primase, is required. Primase synthesises a short stretch of RNA, termed a primer, to initiate DNA synthesis. The RNA primer serves as a starting point for DNA polymerase, which then adds deoxynucleotides to extend it, synthesising the new DNA strand.
DNA replication involves two types of DNA polymerases to overcome the challenge of primer removal and replacement. DNA polymerase III is responsible for extending the RNA primer by adding deoxynucleotides. However, it must detach from the template when it encounters another Okazaki fragment's primer. At this point, another enzyme, DNA polymerase I, takes over. DNA polymerase I removes the RNA nucleotides of the primer and replaces them with DNA nucleotides, filling in the gap left behind by the RNA primer.
Finally, the Okazaki fragments need to be joined into a single continuous DNA strand by the enzyme DNA ligase.
The video below provides a summary of the DNA replication process with some additional details.
