Once initiated, DNA replication proceeds by coordinated copying of both leading and lagging strands. Although both bacteria and eukaryotes have multiple DNA polymerases, only one, named polymerase III (Pol III), is primarily responsible for replicative DNA synthesis in E. coli. In eukaryotes, DNA polymerases δ and ε have both been implicated in this process along with a suggestion that each of these two enzymes may be specific for leading or lagging strand synthesis.
Replication involves separation of two DNA strands which are catalyzed by DNA helicases which hydrolyze ATP during this reaction. ATP hydrolysis provides the energy needed for the unwinding process. All cells have multiple DNA helicases for a variety of DNA transactions. DnaB is the key helicase for replication of the genome E. coli. However, other helicases such as Rep and PriA are also involved in replication and interact with other components of the replication complex called the replisome.
Replication requires a large number of proteins, including the holoenzyme of Pol III which includes, in addition to the catalytic polymerase cores, ten or more pairs of other subunits. The polymerase complex appears to have a dimeric asymmetric structure in order to replicate simultaneously two strands with opposite polarity. The continuous leading strand synthesis should be processive without interruption, because periodic RNA primer synthesis is not necessary once the leading DNA strand synthesis is initiated. On the other hand, the discontinuous lagging strand synthesis should not be processive, because repeated synthesis of RNA primers is required to initiate synthesis of each Okazaki fragment. The Pol III holoenzyme appears to assemble in a stepwise fashion, with its key β-subunit dimer acting as a sliding clamp based on its X-ray crystallographic structure of a ring surrounding the DNA. This clamp is loaded on DNA by the γ -complex, accompanied by ATP hydrolysis. The dimeric structure of the replication complex is maintained by the dimeric subunit of the holoenzyme. The β-clamp slides on the duplex DNA template and thus promotes processivity. Proliferating cell nuclear antigen (PCNA) is the sliding clamp homolog in eukaryotic cells and is also used in SV40 replication.
Much of the information about the composition of the E. coli Pol III holoenzyme, and DNA chain elongation, was generated from studies of the replication of small, single-stranded circular DNAs of bacterial viruses φX174 and M13 and also of laboratory-constructed plasmid DNA containing the ori (ori C) of E. coli. Asymmetric dimeric replication complexes have also been identified for larger E. coli viruses such as T4 with a linear genome and for the mammalian SV40 virus with a double-strand circular genome. In circular genomes, DNA synthesis is terminated at around 180° from the origin. In the case of linear genomes, termination occurs halfway between two neighboring replicons. The mechanism of termination is not completely understood. Although, in the E. coli genome, specific termination (ter) sequences are present, which bind to terminator proteins, such proteins act as anti-helicases to prevent strand separation. However, the termination may not be precise and occurs when the replicating forks collide.
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