Journal of Molecular Biology
CommunicationMcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae
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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2016, Journal of Molecular BiologyCitation Excerpt :It has previously shown that Mcm10 interacts with Cdc45 in in vitro [31]. Furthermore, budding yeast Mcm10 is required for the timely recruitment of Cdc45 to the Mcm2–7 complex during the S phase [31,45]. To determine whether the Mcm10–Cdc45 interaction was disrupted in our mutant, we performed a GST pull-down assay.
The replication initiation protein Sld3/Treslin orchestrates the assembly of the replication fork helicase during S phase
2015, Journal of Biological ChemistryCitation Excerpt :The recruitment of Cdc45 to Mcm2–7 has also been reported to be dependent upon DDK (9, 11, 25–27). Mcm10 has been reported by some laboratories to be important for the recruitment of Cdc45 to Mcm2–7 (28–31), whereas other labs have reported that Mcm10 is not required for Cdc45 recruitment to Mcm2–7 (32–34). A second key event in helicase assembly is the attachment of GINS to the Mcm3 and Mcm5 subunits of the Mcm2–7 complex during S phase (15, 17, 35, 36).
MCM10: One tool for all-Integrity, maintenance and damage control
2014, Seminars in Cell and Developmental BiologyEnigmatic roles of Mcm10 in DNA replication
2013, Trends in Biochemical SciencesCitation 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].
Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS
2012, Current BiologyCitation 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.
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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.