DNA replication in prokaryotes - principles of biology (2023)

The prokaryotic chromosome is a circular molecule with a less extensive coiled structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and tightly wound around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two forms of life. DNA replication in prokaryotes has been studied extensively, so let's learn the basic process of prokaryotic DNA replication and then focus on the differences between prokaryotes and eukaryotes.

How does the replication machinery know where to start? It turns out that there are so-called specific nucleotide sequencesorigins of replicationwhere does replication beginE colihas a single origin of replication on its single chromosome, like most prokaryotes (illustration 1🇧🇷 The origin of replication is approximately 245 base pairs long and is rich in AT sequences. This sequence of base pairs is recognized by specific proteins that bind to this site. An enzyme calledHelicasaunwinds DNA by breaking the hydrogen bonds between pairs of nitrogenous bases. ATP hydrolysis is required for this process as it requires energy. As the DNA unfolds, the Y-shaped structures are calledThe replication forkThey are formed (illustration 1🇧🇷 Two replication forks form at the origin of replication, expanding bidirectionally as replication progresses.single chain binding proteins(Figure 2) Cover the individual DNA strands near the replication fork to prevent the single-stranded DNA from re-forming a double helix.

The next important enzyme isDNA polymerase III, also known as DNA pol III, which adds nucleotides one by one to the growing DNA strand (Figure 2). Adding nucleotides requires energy; This energy is derived from the nucleotides to which three phosphates are attached. Structurally, ATP is an adenine nucleotide to which three phosphate groups are attached; breaking down the third phosphate releases energy. In addition to ATP, there are also TTP, CTP and GTP. Each of them consists of the corresponding nucleotide with three phosphates 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 existing strand.

Three main types of polymerases are known in prokaryotes: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I is used later in the process and DNA pol II is used primarily for repair (this is another annoying example of naming based on the order of discovery rather than the order that makes sense).

DNA polymerase can only add nucleotides in the 5' to 3' direction (a new strand of DNA can only extend in this direction). It requires a free 3'-OH group (located on the sugar) to which you can add the next nucleotide forming a phosphodiester bond between the 3'-OH end and the 5'-phosphate of the next nucleotide. Basically, this means that you cannot add nucleotides if there is no free 3'-OH group available. So how do you add the first nucleotide? The problem is solved with the help of aprimerwhich provides the free 3'-OH end. another enzymePrimase-RNA, synthesizes an RNA primer five to ten nucleotides long and is complementary to DNA. RNA primase does not require a free 3'-OH group. Since this sequence initiates DNA synthesis, it is appropriately called a primer. DNA polymerase can now extend this RNA primer by sequentially adding complementary nucleotides to the template strand (Figure 2).

The replication fork moves at a rate of 1000 nucleotides per second. DNA polymerase can only extend from 5' to 3', which poses a minor problem at the replication fork. As we know, the DNA double helix is ​​antiparallel; that is, one strand is oriented in the 5' to 3' direction and the other in the 3' to 5' direction. A strand complementary to the initial 3' to 5' strand of DNA is continuously synthesized in the direction of the replication fork because the polymerase can add nucleotides in that direction. This continuously synthesized strand is known asmain topic🇧🇷 The other strand, complementary to the parental DNA 5' to 3', extends away from the replication fork in short fragments known asOkazaki-Fragmente, each of which requires an initiator to initiate synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The tape containing the Okazaki fragments is known as Thelate wire.

The leading strand can be extended with a single primer, while the lagging strand requires a new primer for each of the short Okazaki fragments. The general direction of the back strand is 3' to 5' and the main strand is 5' to 3'. A protein calledsliding clampit holds the DNA polymerase in place while continuing to add nucleotides. The slider clamp is a ring-shaped protein that binds to DNA and holds the polymerase in place.topoisomeraseprevents excessive winding of the DNA double helix in front of the replication fork when the DNA is unwrapped; It does this by creating temporary cuts in the DNA helix and then resealing it. As synthesis progresses, the RNA primers are replaced with DNA pol I, which degrades the RNA and fills in the gaps with DNA nucleotides. The enzyme seals the gaps between newly synthesized DNA (replacing the RNA primer) and previously synthesized DNA.DNA-ligasewhich catalyzes the formation of the phosphodiester bond between the 3'-OH end of one nucleotide and the 5'-phosphate end of the other fragment.

(Lisa's note: I find it almost impossible to visualize this process by reading the text. I highly recommend you watch some animations/videos like what's availablehere🇧🇷 There are additional links on Blackboard)

Once the chromosome has fully replicated, the two copies of the DNA move into two different cells during cell division. The process of DNA replication can be summarized as follows:

1. DNA is unwound at the origin of replication.
2. Helicase opens the replication forks that make up DNA; these extend in both directions.
3. Single-stranded binding proteins coat the DNA around the replication fork to prevent DNA rewinding.
4. Topoisomerase binds to the region ahead of the replication fork to prevent supercoiling (supercoiling).
5. Primase synthesizes RNA primers that are complementary to the DNA strand.
6. DNA polymerase III begins adding nucleotides to the 3'-OH (sugar) end of the primer.
7. Stretching of the lagging strand and leading strand continues.
8. RNA primers are removed and gaps filled in with DNA using DNA pol I.
9. The spaces between the DNA fragments are sealed with DNA ligase.

Table 1: Enzymes involved in prokaryotic DNA replication and their respective functions.

DNA replication has been very well studied in prokaryotes, mainly due to the small size of the genome and the large number of available variants.Escherichia colithat's 4.6 million base pairs on a single circular chromosome, and it all replicates in about 42 minutes, starting from a single origin of replication and working its way around the chromosome in both directions. This means that about 1000 nucleotides are added per second. The process is much faster than in eukaryotes.

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OpenStaxName, Concepts of Biology. Open Stax CNX. May 18, 2016http://cnx.org/contents/s8Hh0oOc@9.10:2ousESf0@5/DNA-Replicación