Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins

Abstract

In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G1/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.T he initiation of eukaryotic DNA replication is a multistep process that requires the assembly of the prereplicative complex (pre-RC), activation of the pre-RC and formation of the replisome (1, 2). Pre-RC assembly occurs during the G 1 phase of the cell cycle by a stepwise recruitment of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex onto DNA origins. During the G 1 /S transition stage, the recruitment of other replication factors such as Cdc45 and GINS and the combined actions of two S phase promoting kinases, the cyclindependent (CDK) and Cdc7-Dbf4 (DDK) kinases, lead to the assembly of an active DNA helicase complex at replication origins. This activation results in the unwinding of replication origins, the recruitment of DNA polymerases and accessory factors and the assembly of the replisome for DNA synthesis.In eukaryotic cells, the Mcm2-7 complex seems to be the catalytic core of the replicative helicase. It is essential for origin unwinding and replication fork progression (3-6). It has been shown that a variety of Mcm complexes, including the Mcm4/6/7 subcomplex (from many species), double hexameric complexes of Mcm homologues in Archaea and the budding yeast Mcm2-7 complex, exhibit DNA helicase activity in vitro (1, 7). In vivo, however, formation of an active helicase at replication origins requires the further recruitment of several factors including Cdc45 and GINS, and this activation process is governed by CDK and DDK (1).Both Cdc45 and GINS are required for the establishment and progression of the replication fork (8, 9). These proteins are loaded onto origins during S phase and form a stable complex with Mcm2-7 (10). The complex seems to be a DNA unwinding complex and moves along DNA as part of the replication fork complex (6, 11). Consistent with these observations, a complex of Mcm2-7, Cdc45, and GINS (the CMG complex), purified from D...

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

“…Moreover, the association of RecQ4 with chromatin and the association between TopBP1 and the RecQ4 amino-terminal domain do not require CDK (Sangrithi et al 2005;Matsuno et al 2006). Furthermore, the formation of the CMG complex in HeLa cells requires RecQ4 and Mcm10 (Im et al 2009), and RecQ4 expressed in human 293 cells binds to Mcm10 and associates with the CMG complex in an Mcm10-dependent manner (Xu et al 2009). These results suggest that RecQ4 functions later in the initiation step compared with Sld2.…”

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

“…Interactions between TopBP1-interacting, replication-stimulating protein (Treslin) (homolog of Sld3) and MDM two binding protein (MTBP) (homolog of Sld7) with topoisomerase-IIbeta-binding protein 1 (TopBP1) (homolog of Dpb11) have been detected in higher eukaryotes, suggesting that the mechanism used by yeast for the chromatin loading of Cdc45 and GINS may be conserved (8). However, in vivo studies in HeLa cells using bimolecular fluorescence complementation assays reported that Mcm10 as well as RecQ protein-like 4 (RecQL4) (homolog of Sld2) and Ctf4 are required for human CMG (hCMG) complex formation (9).…”

And‐1 coordinates with Claspin for efficient Chk1 activation in response to replication stress

“…Previous studies by others and us showed that the acidic nucleoplasmic DNA-binding protein 1 (And-1) is a replisome component and is required for efficient DNA replication in unperturbed cells (Zhu et al, 2007;Errico et al, 2009;Gambus et al, 2009;Im et al, 2009;Yoshizawa-Sugata & Masai, 2009;Bermudez et al, 2010;Li et al, 2012b). Ctf4/Mcl1, the ortholog of And-1 in yeast, is required for chromosome transmission fidelity, sister chromatin cohesion, DNA damage repair, maintenance of genome integrity, and the regulation of telomere replication (Kouprina et al, 1992;Hanna et al, 2001;Williams & McIntosh, 2002;Mayer et al, 2004;Tsutsui et al, 2005).…”