User:Mbarnett2014/MG article

Prokaryotic DNA replication
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.

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 9bp 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
With the origin primed, DnaG releases DnaC from the DUE and DNA Pol III is loaded on to begin its work; this marks the start of elongation, which will continue bidirectionally.

The structures associated with elongation are helicase, DNA polymerase, the sliding clamp (and the loader for the lagging strand ), primase, DNA ligase, and several topoisomerases.

DNA polymerases synthesize DNA along a template strand, starting from a primer, in the direction of 5' toward 3'. Other DNA polymerases serve other functions, such as replacing RNA primers with DNA.

For replication to continue, the DNA strands have to be unlinked and unwound, which twists the rest of the chromosome. Various forms of topoisomerase will cut the DNA to relieve this stress and ultimately separate the two new chromosomes after termination.

Helicase unwinds the DNA by wrapping around the duplex and separating them into individual strands until it comes to a termination sequence.

Ligase fuses the lagging strand together.

Primase assembles RNA primers on the template strand, from which the polymerase can then extend the new strand ,

The sliding clamp wraps around the DNA while holding the polymerase in place. Its ring shape means a loader must open it to place it onto the DNA right where the polymerase will be loaded.

Replication restart/DNA repair

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.