Summary Mcm2-7 helicase is loaded onto double stranded origin DNA as an inactive double hexamer (DH) in G1 phase. The mechanisms of Mcm2-7 remodeling to trigger helicase activation in S phase remain unknown. Here, we develop an approach to detect and purify the endogenous DHs directly. Through cellular fractionation, we provide in vivo evidence that DHs are assembled on chromatin in G1 phase and separated during S phase. Interestingly, Mcm10, a robust MCM interactor, co-purifies exclusively with the DHs in the context of chromatin. Deletion of the main interaction domain, Mcm10 C-terminus, causes growth and S phase defects, which can be suppressed through Mcm10-MCM fusions. By monitoring the dynamics of MCM DHs, we show a significant delay in DH dissolution during S phase in the Mcm10-MCM interaction deficient mutants. Therefore, we propose an essential role for Mcm10 in Mcm2-7 remodeling through formation of a cell cycle regulated supercomplex with DHs.

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-K complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division.

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DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa.

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