Communication
Mcm10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae

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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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    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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