Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Abstract

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...

Loss of GINS2 inhibits cell proliferation and tumorigenesis in human gliomas

“…To determine the possible mechanism by which GINS2 inhibits glioma growth, key molecules in signaling pathways regulating the cell cycle were assessed. The GINS2 gene and MCM2 molecule jointly participate in the process of DNA replication by forming the “minimum complex.” The detection of MCM2 and ATM proteins in the Input group revealed normal expression. The detection of MCM2 protein in the IP group suggested that Co‐IP acquired MCM2, the bait protein; the detection of ATM protein highlighted the interaction between ATM and MCM2 (Figure A).…”

Mechanisms and regulation of DNA replication initiation in eukaryotes

“…Each MCM2-7 binds a single copy of Cdc45 and one GINS tetramer to form a stable, eleven-protein complex termed the CMG (for C dc45• M CM2-7• G INS). The CMG has been shown to possess robust helicase activity (Moyer et al, 2006, Ilves et al, 2010), and bioinformatics analyses have indicated that a full set of CMG factors are likely conserved from archaea to eukaryotes (Makarova et al, 2012), with recent work demonstrating that, as observed for the eukaryotic accessory factors, archaea’s Cdc45 and GINS homologs activate MCM in vitro and in vivo (Xu et al, 2016). …”

Replication fork pausing at protein barriers on chromosomes

“…The key mechanism for establishing a stable association of replisomes with the replication forks is the maintenance of DNA in the hole of the ring‐shaped structure of replicative helicases. Replicative helicases, including gp4 in bacteriophage T7 , DnaB in Escherichia coli , MCM homo‐hexamer–Cdc45/RecJ‐like protein–GINS complex in archaea , and Cdc45, Mcm2–7 hexamer and GINS complex (CMG complex) in eukaryotes , have a ring‐like shape and are considered to hold unwound ssDNA in the hole of the ring . The helicases bound stably to DNA associate with DNA polymerase, which also binds to unwound ssDNA for DNA synthesis and interacts with a clamp complex, the β clamp in E. coli , or PCNA in eukaryotes.…”