Prokaryotic DNA replication

Prokaryotic DNA Replication
DNA replication in E. coli is bi-directional and originates at a single origin of replication (OriC). The initiation of replication is mediated by DnaA, a protein that binds to a region of the origin known as the DnaA box. In E. coli, there are 5 DnaA boxes, each of which contains a highly conserved 9bp consensous sequence 5' - TTATCCACA - 3'. Binding of DnaA to this region causes it to become negatively supercoiled. Following this, a region of OriC upstream of the DnaA boxes (known as DnaB boxes) become melted. There are three of these regions, and each are 13bp long, and AT rich (which obviously facilitates melting because less energy is required to break the two hydrogen bonds that form between A and T nucleotides). This region has the consensous sequence 5' - GATCTNTTNTTTT - 3. Melting of the DnaB boxes requires ATP (which is hydrolyzed by DnaA). Following melting, DnaA recuits a hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. This is where the replication fork will form. Recruitment of helicase requires six DnaC proteins, each of which is attached to one subunit of helicase. Once this complex is formed, an additional five DnaA proteins bind to the original five DnaA proteins to form five DnaA dimers. DnaC is then released, and the prepriming complex is complete. In order for DNA replication to continue, SSB is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing, and DNA gyrase is needed to relieves the stress (by creating negative supercoils) created by the action of DnaB helicase. The unwinding of DNA by DnaB helicase allows for primase (DnaG) and RNA polymerase to prime each DNA template so that DNA synthesis can begin.

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 deoxynucleotides 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. Nucleophillic attack by the 3' OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed (by inorganic phosphatase) into two phophates. 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 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 in the active site has a wider 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' and the other is 5' -> 3'. To solve this replication in opposite directions. Heading towards the replication fork, the leading strand in 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 RNAseH and DNA Polymerase I (exonuclease), and the gap (or nick's) are filled with deoxyribonucleotides and sealed by the enzyme ligase.

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. However, these sequences are not required for termination of replication.

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 hemimethylated GATC DNA sequences. This four bp sequences occurs 11 times in OriC, and newly synthesized DNA only has its parent strand methylated. DAM methyltrasferase methylates the newly synthesized strand of DNA ONLY if it is not bound to SeqA. The importance of hemimethylation is twofold. Firstly, OriC becomes inaccessible to DnaA, and secondly, DnaA binds better to fully methylated DNA than hemimethylated DNA.