Mechanisms for initiating cellular DNA replication

Abstract

The initiation of DNA replication represents a committing step to cell proliferation. Appropriate replication onset depends on multiprotein complexes that help properly distinguish origin regions, generate nascent replication bubbles, and promote replisome formation. This review describes initiation systems employed by bacteria, archaea, and eukaryotes, with a focus on comparing and contrasting molecular mechanisms among organisms. Although commonalities can be found in the functional domains and strategies used to carry out and regulate initiation, many key participants have markedly different activities and appear to have evolved convergently. Despite significant advances in the field, major questions still persist in understanding how initiation programs are executed at the molecular level.

Figure 1

Overview of origin organization. (a) Bacterial oriCs from three representative species (Escherichia coli, Bacillus subtilis, Streptomyces lividans). Sites bound by DnaA and other factors, as well as the DNA-unwinding element (DUE), are labeled. (b) Saccharomyces cerevisiae ARS1 (autonomous replicating sequence 1). ARS consensus sequence elements (A, B1, B2) and ORC · Cdc6 binding sites are shown. (c) The three origins of Sulfolobus solfataricus. DUEs and binding sites for replication initiators are labeled. Abbreviations: ORB, origin recognition box; ORC, origin recognition complex; WhiP, winged-helix initiator protein.

Figure 2

Structure and mechanism of DnaA. (a) Domain architecture of DnaA. Protein Data Bank models for domain I and an ADP state of domains III/IV of DnaA are shown (7, 62, 151). Inset: structure of domain IV bound to duplex DNA (28). (b) Structure of an AMPPCP-assembled DnaA helix (domains III and IV) bound to an extended single-stranded DNA substrate (magenta) (72). (c) Two-state model for IHF-assisted DnaA melting of oriC (7). This mechanism accounts for the existence of two different DnaA oligomers (12, 74), one of which wraps the organizing center of oriC and another that melts the DNA-unwinding element (DUE). (d) Loopback model for IHF-assisted melting, in which a single DnaA complex bound at the organizing center of oriC opens the DUE (296).

Figure 3

Structure of origin recognition complex (ORC)-type initiators. (a) Schematic of domains in Orc proteins. (b) Three-dimensional electron microscopy (EM) model of the Drosophila ORC indicating where the AAA+ (ATPases associated with various cellular activities) domains of the complex reside (89). (c) Three-dimensional EM model of the budding yeast ORC · Cdc6 complex. The positions of AAA+ subunits are labeled (103). (d) Structures of archaeal Orc1 proteins show variability in AAA+/WHD (winged-helix domain) position [Pyrobaculum aerophilum (4); Sulfolobus solfataricus (116)]. (e) Structure of S. solfataricus Orc1-1 and Orc1-3 paralogs complexed with overlapping sites in Sso-oriC2 (116). Abbreviation: BAH, bromo-adjacent homology.

Figure 4

DnaB structure and loading. (a) Closed-ring DnaB hexamer from Bacillus stearothermophilus (128). (b) Structure of a spiral DnaC oligomer [AAA+ (ATPases associated with various cellular activities) domains] bound to nucleotide (147). (c) Model for loading of DnaB onto oriC following the two-state mechanism shown in Figure 1c (147). Abbreviations: CTD, C-terminal domain; HTH, helix-turn-helix; NTD, N-terminal domain.

Figure 5

Minichromosome maintenance (MCM) structure and loading. (a) A closed-ring MCM homohexamer from Sulfolobus solfataricus (178). (b) Three-dimensional electron microscopy (EM) model for an open Mcm2–7 heterohexamer from Drosophila melanogaster (170). (c) Model for the loading of an Mcm2–7 double hexamer onto DNA by ORC · Cdc6 and Cdt1 (84, 175, 206). (d) Three-dimensional EM model for the Methanothermobacter thermautotrophicus MCM (235) showing a crystallographic model for an N-terminal domain (NTD) dodecamer (171) docked into the central region. (e) Three-dimensional EM model for the D. melanogaster CMG showing how GINS · Cdc45 latch onto and close an Mcm2–7 ring (170). (f) Model for the loading of archaeal MCMs onto DNA by Orc1 (54, 59).

Figure 6

Overview of initiation factor regulation in cells. (a) Regulation of DnaA in bacteria, comparing and contrasting Escherichia coli and Bacillus subtilis systems. (b) Regulation of initiation proteins in eukaryotes. Arrows, activating interactions/events; inhibition lines, repressive interactions/events; hatched text, degradation. Abbreviations: CDK, cyclin-dependent kinase; DARSs, DnaA-reactivating sequences; DDK, Dbf4-dependent kinase; ORC, origin recognition complex; RC, replication complex.