Mcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae

doi:10.1016/j.jmb.2004.04.066

Journal of Molecular Biology

Volume 340, Issue 2, 2 July 2004, Pages 195-202

https://doi.org/10.1016/j.jmb.2004.04.066 Get rights and content

Abstract

Mcm10 has recently been found to play a crucial role in multiple steps of the DNA replication initiation process in eukaryotes. Here, we have examined the role of Mcm10 in assembling initiation factors at a well-characterized yeast replication origin, ARS1. We find that the pre-replication complex (pre-RC) components Cdc6 and Mcm7 associate with ARS1 in the mcm10-1 mutant, suggesting that establishment of the pre-RC is not compromised in this mutant. Association of Cdc45 with ARS1 is reduced in the mcm10-1 mutant, suggesting that Mcm10 is involved in recruiting Cdc45 to the pre-RC. We find that overexpression of either Mcm10-1 or Cdc45 suppresses the growth defect of mcm10-1, and that a physical interaction between Cdc45 and Mcm10 is disrupted in the mcm10-1 mutant. Our results show that interaction between the Mcm10 and Cdc45 proteins facilitates the recruitment of Cdc45 onto the ARS1 origin.

Section snippets

Pre-RC components are loaded onto ARS1 in the mcm10-1 mutant

Mcm10 interacts physically with the MCM2-7 complex⁶ and ORC,¹¹ and so we examined the role of Mcm10 in pre-RC establishment. Utilizing a well-characterized mcm10-ts mutant strain, mcm10-1 (P269L),5., 6. we designed a protocol to assess the formation of pre-RCs at the ARS1 origin using chromatin immunoprecipitation (ChIP) (Figure 1(a)). Cells were first arrested in hydroxyurea (HU), then released to nocodozole to arrest cells at the very beginning of mitosis (Noc sample). This culture was

Cdc45 is not efficiently recruited onto ARS1 in the mcm10-1 mutant

We next examined the role of Mcm10 in recruiting Cdc45. Recent studies in X. laevis and S. pombe showed that Mcm10 is essential for the recruitment of Cdc45 onto chromatin,7., 9. but its role in the recruitment of Cdc45 specifically to origins has not been examined.

We used ChIP to assay the recruitment of Cdc45 to ARS1 in the mcm10-1 mutant. Because Cdc45 is not recruited to origin DNA until the onset of S phase, we designed a protocol where cells traverse the G1–S transition at either the

Cooperation between Cdc45 and Mcm10 involves physical interaction of the two proteins

The results in the previous Figure indicate that Mcm10 is involved in the recruitment of Cdc45 to a replication origin. We find that increased dosage of Cdc45 suppresses the temperature-sensitive growth defect of mcm10-1 (Figure 3(a)). Wild-type cells, but not mcm10-ts mutants, containing an empty vector are able to grow at the restrictive temp of 36 °C (Figure 3(a), (1)). CDC45 was cloned on a plasmid downstream of the GAL1 promoter (pRS313Gal1-CDC45), or into a 2μ vector (pRS423-CDC45).

Acknowledgments

We thank Bruce Stillman for yeast strains and Jose Tercero for advice on antibody conditions. We thank Ming Lei, Rey Chen, Justin Donato, and Tim Christensen for critical reading of this manuscript. This work was supported by NIH grants (GM34190) to B.K.T. and (5 T32 GM007617) to S.L.S.

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The Mcm2–7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing
2017, Journal of Biological Chemistry
Citation Excerpt :
Additionally, its exact contribution to chromosomal DNA replication is still controversial, because there are multiple discrepancies among studies on Mcm10. Studies in budding yeast, fission yeast, Xenopus, Drosophila, and humans have shown that Mcm10 is required for the recruitment of Cdc45 to origins and formation of the CMG complex (37–41). The chromatin loading of Mcm10 is independent of Cdk2 and Cdc7 in a Xenopus egg extract system (37).
The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2–7-interacting domain in human Mcm10. The interaction with Mcm2–7 required the Mcm10 domain that contained amino acids 530–655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200–482) is essential for DNA replication, whereas both the conserved and the Mcm2–7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2–7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.
An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation
2016, Journal of Molecular Biology
Citation Excerpt :
However, this disruption must be transient, since Mcm10 remains associated with Mcm2–7 throughout the S phase [30,31]. Mcm10 binds and recruits Cdc45 to Mcm2–7 in an in vitro assay [31,45]. Furthermore, ssDNA and bubble DNA, but not dsDNA, also disrupt the interaction between Mcm10 and Cdc45 (Fig. 1f, g, and h).
Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2–7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2–7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS–Mcm2–7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication.
The replication initiation protein Sld3/Treslin orchestrates the assembly of the replication fork helicase during S phase
2015, Journal of Biological Chemistry
The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2–7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2–7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2–7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.
MCM10: One tool for all-Integrity, maintenance and damage control
2014, Seminars in Cell and Developmental Biology
Citation Excerpt :
The Masukata laboratory has provided some evidence that Mcm10's ability to bind ssDNA is important in this context [27]. It is likely however, that additional properties of Mcm10 make it the ideal tool for this task, as it has been shown to interact with Cdc45 [16,30,31], distinct subunits of Mcm2-7 and GINS [2,20,30,32–34], ORC [30,33–35], the Sld2 ortholog Recql4 [36] and the ssDNA binding complex replication protein A (RPA) [5]. Although RPA has a 40-fold higher affinity for ssDNA than Mcm10 [13], the latter also binds dsDNA [10,12,13], uniquely enabling it to bind parental and unwound DNA.
Minichromsome maintenance protein 10 (Mcm10) is an essential replication factor that is required for the activation of the Cdc45:Mcm2-7:GINS helicase. Mcm10's ability to bind both ds and ssDNA appears vital for this function. In addition, Mcm10 interacts with multiple players at the replication fork, including DNA polymerase-α and proliferating cell nuclear antigen with which it cooperates during DNA elongation. Mcm10 lacks enzymatic function, but instead provides the replication apparatus with an oligomeric scaffold that likely acts in the coordination of DNA unwinding and DNA synthesis. Not surprisingly, loss of Mcm10 engages checkpoint, DNA repair and SUMO-dependent rescue pathways that collectively counteract replication stress and chromosome breakage. Here, we review Mcm10's structure and function and explain how it contributes to the maintenance of genome integrity.
Enigmatic roles of Mcm10 in DNA replication
2013, Trends in Biochemical Sciences
Citation Excerpt :
Early studies in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Xenopus laevis egg extracts suggested that Mcm10 was loaded onto chromatin after origin licensing but before the initiation of DNA synthesis [4,11,12]. Subsequent work in multiple different model organisms revealed a potential role for Mcm10 in the recruitment of the helicase coactivator Cdc45 [4,12,13]. As alluded to above, this view was recently challenged by in vitro experiments with budding yeast whole cell extracts, which demonstrated that the CMG complex assembled in the absence of Mcm10 [8].
Minichromosome maintenance protein 10 (Mcm10) is required for DNA replication in all eukaryotes. Although the exact contribution of Mcm10 to genome replication remains heavily debated, early reports suggested that it promotes DNA unwinding and origin firing. These ideas have been solidified by recent studies that propose a role for Mcm10 in helicase activation. Whereas the molecular underpinnings of this activation step have yet to be revealed, structural data on Mcm10 provide further insight into a possible mechanism of action. The essential role in DNA replication initiation is not mutually exclusive with additional functions that Mcm10 may have as part of the elongation machinery. Here, we review the recent findings regarding the role of Mcm10 in DNA replication and discuss existing controversies.
Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS
2012, Current Biology
Citation Excerpt :
These results support the hypothesis that Mcm10 plays a role independent of CMG formation. A previous report showed that Cdc45 association with ARS1 is partially reduced after releasing the mcm10-1 cells in HU medium for 90 min at 36°C [7]. Because Cdc45 association with origins gradually declined following the time course in the absence of Mcm10 (Figure 3B, −Mcm10), the former analysis might have caught the declined association signal of Cdc45 at a later time point.
Eukaryotic DNA replication is initiated at multiple origins of replication, where many replication proteins assemble under the control of the cell cycle [1]. A key process of replication initiation is to convert inactive Mcm2–7 to active Cdc45-Mcm-GINS (CMG) replicative helicase [2]. However, it is not known whether the CMG assembly would automatically activate its helicase activity and thus assemble the replisome. Mcm10 is an evolutionally conserved essential protein required for the initiation of replication [3, 4]. Although the roles of many proteins involved in the initiation are understood, the role of Mcm10 remains controversial [5, 6, 7, 8, 9]. To characterize Mcm10 in more detail, we constructed budding yeast cells bearing a degron-fused Mcm10 protein that can be efficiently degraded in response to auxin. In the absence of Mcm10, a stable CMG complex was assembled at origins. However, subsequent translocation of CMG, replication protein A loading to origins, and the intra-S checkpoint activation were severely diminished, suggesting that origin unwinding is defective. We also found that Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner. Thus, Mcm10 plays an essential role in functioning of the CMG replicative helicase independent of assembly of a stable CMG complex at origins.

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^†: Present addresses: Sara L. Sawyer, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Irene H. Cheng, Gladstone Institute of Neurological Disease, UCSF, San Francisco, CA 94141, USA; Weihang Chai, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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Journal of Molecular Biology

CommunicationMcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae

Abstract

Section snippets

Pre-RC components are loaded onto ARS1 in the mcm10-1 mutant

Cdc45 is not efficiently recruited onto ARS1 in the mcm10-1 mutant

Cooperation between Cdc45 and Mcm10 involves physical interaction of the two proteins

Acknowledgments

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Methods Enzymol.

DNA replication in eukaryotic cells

Annu. Rev. Biochem.

Initiating DNA synthesis: from recruiting to activating the MCM complex

J. Cell Sci.

Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase

Mol. Cell. Biol.

Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins

Genes Dev.

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae

Mol. Cell Biol.

Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding

Mol. Biol. Cell

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase

Nucl. Acids Res.

Communication
Mcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae