Prokaryotic DNA replication is the process by which a prokaryote duplicates its entire genome into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initation, elongation, and termination.
Contents
Initiation
DNA replication begins at the origin of replication, a region commonly containing repeating sequences (DnaA boxes) that bind DnaA, an initiation protein. DnaA-ATP will first bind high-affinity boxes (R1, R2, and R4, which have a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'), then oligomerize into several low-affinity boxes. This accumulation will displace a protein called Fis, allowing another protein, IHF, to bind the DNA and induce a bend. This allows the DnaA chain to load onto an AT-rich region of 13-mers (the DUE, Duplex unwinding element), causing the double-stranded DNA to separate. The DnaC helicase loader will interact with the DnaA on the single-stranded DNA to recruit the DnaB helicase, which will continue to unwind the DNA as the DnaG primase lays down an RNA primer and DNA Polymerase III holoenzyme begins elongation.
Regulation
Chromosome replication in bacteria is regulated at the initiation stage. DnaA-ATP is hydrolyzed into the inactive DnaA-ADP by RIDA (Regulatory Inactivation of DnaA), and converted back to the active DnaA-ATP form by DARS (DnaA Reactivating Sequence, which is itself regulated by Fis and IHF). However, the main source of DnaA-ATP is synthesis of new molecules. Meanwhile, several other proteins interact directly with the oriC sequence to regulate initiation, usually by inhibition. In E. coli these proteins include DiaA, SeqA, IciA, HU, and ArcA-P, but they vary across other bacterial species. A few other mechanisms in E. coli that variously regulate initiation are DDAH (datA-Dependent DnaA Hydrolysis, which is also regulated by IHF), inhibition of the dnaA gene (by the SeqA protein), and reactivation of DnaA by the lipid membrane.
Elongation
Once priming is complete, DNA polymerase III holoenzyme is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase III involves the use of two metal ions in the active site, and a region in the active site that can discriminate between deoxyribonucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3' OH initiate a nucleophilic attack onto the alpha phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3' OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed (by inorganic phosphatase) into two phosphates. This hydrolysis drives DNA synthesis to completion.
Furthermore, DNA polymerase III must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, dsDNA (double stranded DNA) in the active site has a wider major groove and shallower minor groove that permits the formation of hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions.
DNA is read in the 3' → 5' direction, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' → 3' direction. However, one of the parent strands of DNA is 3' → 5' while the other is 5' → 3'. To solve this, replication occurs in opposite directions. Heading towards the replication fork, the leading strand is synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand, heading away from the replication fork, is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNase H and DNA Polymerase I (exonuclease), and the gaps (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.
Rate of replication
The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli. During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.
Termination
Termination of DNA replication in E. coli is completed through the use of termination sequences and the Tus protein. These sequences allow the two replication forks to pass through in only one direction, but not the other.
DNA replication initially produces two catenated or linked circular DNA duplexes, each comprising one parental strand and one newly synthesised strand (by nature of semiconservative replication). This catenation can be visualised as two interlinked rings which cannot be separated. Topoisomerase IV in E. coli unlinks or decatenates the two circular DNA duplexes.
Regulation
Regulation of DNA replication is achieved through several mechanisms. Mechanisms involve the ratio of ATP to ADP, of DnaA to the number of DnaA boxes and the hemimethylation and sequestering of OriC.
The ratio of ATP to ADP indicates that the cell has reached a specific size and is ready to divide. This "signal" occurs because in a rich medium, the cell will grow quickly and will have a lot of excess DNA.
Furthermore, DnaA binds equally well to ATP or ADP, and only the DnaA-ATP complex is able to initiate replication. Thus, in a fast-growing cell, there will be more DnaA-ATP than DnaA-ADP. Because the levels of DnaA are strictly regulated, and 5 DnaA-DnaA dimers are needed to initiate replication, the ratio of DnaA to the number of DnaA boxes in the cell is important. After DNA replication is complete, this number is halved, thus DNA replication cannot occur until the levels of DnaA protein increases.
Finally, DNA is sequestered to a membrane-binding protein called SeqA. This protein binds to hemi-methylated GATC DNA sequences. This four bp sequences occurs 11 times in OriC, and newly synthesized DNA only has its parent strand methylated. DAM methyltransferase methylates the newly synthesized strand of DNA only if it is not bound to SeqA. The importance of hemi-methylation is twofold. Firstly, OriC becomes inaccessible to DnaA, and secondly, DnaA binds better to fully methylated DNA than hemi-methylated DNA.
These mechanisms serve to downregulate DNA replication so that it only occurs once per cell cycle, preventing over-replication of DNA.