Bacterial and Eukaryotic Replisome Machines

Abstract

Cellular genomic DNA is replicated by a multiprotein replisome machine. The replisome contains numerous essential factors that unwind, prime and synthesize each of the two strands of duplex DNA. The antiparallel structure of DNA, and unidirectional activity of DNA polymerases, requires the two strands of DNA to be extended in opposite directions, and this structural feature requires distinctive processes for synthesis of the two strands. Genome duplication is of central importance to all cell types, and one may expect the replisome apparatus to be conserved from bacteria to human, as is the case with RNA polymerase driven transcription and ribosome mediated translation. However, it is known that the replication factors of bacteria are not homologous to those of archaea and eukaryotes, indicating that the replication process evolved twice, independently, rather than from a common ancestor cell. Thus, the different domains of life may exhibit significant differences in their mechanistic strategy of replication. In this review, we compare and contrast the different structures and mechanistic features of the cellular replication machinery in the three domains of life.

Keywords: CMG; DNA helicase; DNA polymerase; Primase; Replisome.

Figure 1

Clamps and clamp loaders. a) The beta clamp of E. coli is a homodimer that consists of 6 globlular domains (pdb 2POL). b) The eukaryotic PCNA clamp is a homotrimer that consists of 6 domains (1AXC). Structure (panel c) and illustration (panel d) of the T4 clamp loader bound to an open clamp (grey) and DNA (yellow) (3U60). Panels c and d are reproduced with permission from Figure (2a) of [12].

Figure 2

Proposed translocation mechanisms of hexameric helicases. Replicative helicases are hexameric rings composed of an N-tier and C-tier; the motor domains are in the C-tier. a) Inchworm mechanism. Left: the C- and N-tiers are parallel and compact. Middle: The C-tier opens at one interface and expands into a spiral structure, melting DNA and leaving the N-tier a flat uninterrupted ring. Right: The C-tier reassumes the compact shape and translocates the N-tier on DNA. b) Rotary model. Left: The C-tier is shaped as a spiral lock washer and connects to a flat N-tier ring. Middle: ATP hydrolysis translates the lock washer shape by one protomer, resulting in DNA translocation. Right: ATP hydrolysis translates the lock washer shape by one protomer, resulting in DNA translocation.

Figure 3

Comparison of replisomes from bacteria and eukaryotes. a) The bacterial replisome is organized by a central clamp loading machine (beige) that has C-terminal extension, which extrude from the top and bind the hexameric helicase (blue) that encircles the lagging strand. The clamp loader extensions also bind three copies of Pol III (green), the replicative DNA polymerases. One Pol III functions on the continuous leading strand, tethered to DNA by the beta clamp (yellow). The other two Pol III molecules extend lagging strand fragments that are initiated by short RNA primers synthesized by primase (pink). b) The eukaryotic replisome is organized by the CMG helicase (blue), which functions with Pol epsilon (red) on the leading strand and bind Pol alpha-primase (green) through a Ctf4 trimer (light yellow) on the lagging strand. Lagging strand primers are extended by Pol delta (grey). Both Pol epsilon and Pol delta function with a PCNA clamp (yellow). Whether the RFC clamp loader and Pol delta directly connect to other replisome proteins is not known. Other factors that move with replisomes are shown within the dashed outline; their exact connection points are not yet known.