The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS • Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.T he initiation of DNA replication is an important control point in the progression of the cell cycle. In eukaryotes, origins are defined by the initiator protein ORC complex that, via the actions of two additional factors, the helicase coloaders Cdc6 and Cdt1, directs loading of the MCM(2-7) replicative helicase onto doublestranded DNA. Activation of the MCM(2-7) replicative helicase occurs subsequent to recruitment, leading to DNA melting and assembly of the full replisome apparatus (1, 2). Key steps in MCM activation involve a series of phosphorylation-dependent events that promote the sequential association of Cdc45 and the GINS complex with the chromatin-associated MCM double hexamer. These recruitment events are regulated by the CDK and DDK kinases and require the additional accessory factors Sld3/7, Dpb11, and Sld2(3-6). The Cdc45•MCM(2-7)•GINS complex (CMG) forms the core of the eukaryotic replisome, and this 11-subunit assembly appears to be the functional helicase driving fork progression (3-6). The archaeal replication machinery resembles an ancestral form of its eukaryotic counterpart. Archaea possess a simple homohexameric MCM (5, 7). In addition, archaeal homologs of GINS and Cdc45 have been identified (8)(9)(10)(11)(12)(13)(14). In species of the genus Sulfolobus, we have previously demonstrated that the GINS complex interacts with the N-terminal domains of MCM (8). In Sulfolobus, GINS is a dimer of dimers: one subunit, Gins23, is related to the eukaryotic GINS components Psf2 and Psf3, and the second Sulfolobus subunit, Gins15, is related to the eukaryotic Sld5 and Psf1. These sequence relationships have been confirmed by structural studies of the Thermococcus kodakarensis GINS complex that have demonstrated the tetrameric assembly of archaeal (Gins15) 2 • (Gins23) 2 and validated the organizational similarity of the archaeal and eukaryotic GINS complexes (15). Interestingly, Sulfolobus GINS copurifies over the course of eight steps with a further polypeptide that we initially named RecJdbh, based on its observed homology with the presumptive DNA binding domain of the bacterial exonuclease, RecJ (8). Subsequent sequence analyses have revealed a relationship between RecJ and eukaryotic Cdc45, and this has been elegantly confirmed by recent structural studies of eukaryotic Cdc45 (9,11,16,17). We therefore propose renaming RecJdbh as Cdc45. As archaea lack orthologs of Sld2, Sld...

DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNApolymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.