Further work in Thomas J. Kelly's and other laboratories led to the identification and characterization of each of the cellular proteins required for SV40 DNA replication. The replication machinery from a large number of different eukaryotes, ranging from yeast to humans has now been studied. These studies show that the components of the replication machinery in the cell nucleus are very similar throughout the eukaryotic domain. Therefore, the discussion that follows describes the eukaryotic elongation stage in general. Our task of studying the eukaryotic replication elongation stage is simplified by the fact that the key eukaryotic proteins that participate during this stage work in the same way as their bacterial counterparts.
Leading and lagging strand synthesis are coordinate processes in eukaryotic DNA replication just as they are in bacterial DNA replication. For convenience, we will consider the two processes separately. Leading strand synthesis begins after Pol α completes initiator DNA synthesis. A second polymerase, DNA polymerase δ (Pol δ) elongates initiator molecules synthesized by Pol α. The crystal structure for DNA polymerase δ has not yet been determined. Mammalian Pol δ consists of a large subunit that has both 5'→3'DNA polymerase activity and 3'→5'exonuclease activity and a small subunit with a function that has yet to be determined. Pol δ has a low processivity value. However its processivity becomes quite high when it is tethered to DNA by a sliding clamp. Because the eukaryotic sliding clamp was first detected as an antigen in proliferating cells before its function was known, it was called proliferating cell nuclear antigen (PCNA). This name is still used but the more descriptive name of sliding clamp appears to be gaining in popularity. The eukaryotic sliding clamp has a very similar quaternary structure to that described for the bacterial sliding clamp.
This similarity is remarkable because the protein subunits in the eukaryotic and bacterial sliding clamps are not homologous and the eukaryotic clamp has three subunits as compared to two subunits in the bacterial sliding clamp.
The eukaryotic clamp loader couples ATP hydrolysis to the assembly of the PCNA sliding clamp onto a primed template. Pol δ associates with PCNA for processive DNA synthesis. The process is called polymerase switching because pol δ · PCNA complex (pol δ holoenzyme) displaces Pol α. It needs to take place just once for leading strand synthesis because once chain extension starts, Pol δ remains tethered to the leading strand template. Two protein switches take place during ploymerase switching. Each is driven by a competition-based protein exchange in which two proteins compete for interaction with a third protein.
RPA plays a central role in both switching steps. Pol α must associate with RPA to remain stably attached to its primed site. The first protein switch occurs when replication factor C (RFC), the eukaryotic clamp loader, binds to RPA, disrupting the Pol α ·RPA contact site and causing Pol α to be released. Once the primed site becomes available, RFC loads the PCNA ring onto it. The second protein switch occurs when Pol δ displaces RFC. However RFC is not released but instead establishes new contacts with the Pol δ holoenzyme.
Let us now shift focus to lagging strand synthesis. This process can be divided into two stages, Okazaki fragment synthesis and Okazaki fragment maturation. As the replication machinery moves, the replication bubble continues to grow and Pol α binding sites become available on the lagging strand template. Pol α synthesizes initiator DNA. Then a switch occurs in which Pol δ holoenzyme replaces pol α. The switching process is identical to that described for the leading strand. Subsequent steps in lagging strand synthesis are similar to those described in bacteria except that the Okazaki fragments in mammalian cells are about one-tenth the size of those in bacteria.
