A Novel Zinc Finger Is Required for Mcm10 Homocomplex Assembly

Cook, Craig R.; Kung, Guosheng; Peterson, Francis C.; Volkman, Brian F.; Lei, Ming

doi:10.1074/jbc.m306049200

Cited by 36 publications

(42 citation statements)

References 49 publications

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“…Dimerization of xMcm10-NTD-Purified scMcm10 and spMcm10 have been reported to oligomerize in solution (8,29,31), and human Mcm10 was recently reported to form hexameric assemblies (41). Before a rigorous analysis of xMcm10 oligomerization, we first investigated the hydrodynamic properties of the full-length, NTD, ID, and CTD proteins by gel filtration chromatography (supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Mcm10 from Metazoa contains ϳ100 -300 residues not present in the yeast proteins, and conservation from yeast to human is limited to ϳ200-amino acids in the middle of the protein. Consistent with Mcm10 DNA binding activity, the conserved central region contains an invariant CCCH zinc binding motif (22,23,31) and a putative oligonucleotide/oligosaccharide binding fold (27).…”

mentioning

confidence: 87%

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Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

Robertson

Warren

Zhang

et al. 2008

Journal of Biological Chemistry

125

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Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single-and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase ␣-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase ␣-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.Eukaryotic DNA replication is carried out by large multiprotein machines that coordinate DNA unwinding and synthesis at the replication fork. Initiation of replication involves ordered assembly of the replisome and local denaturation of duplex DNA at the origin followed by replisome activation. Screens for mutants defective in minichromosome maintenance (Mcm) 4 and DNA replication in yeast identified a number of factors essential for replication (1-4). Pre-replicative complexes composed of the origin recognition complex, Cdc6, Cdt1, and the hexameric Mcm2-7 helicase are assembled in G 1 (for review, see Ref. 5) and converted into active replication forks at the onset of S phase. Mcm10 loads onto chromatin after pre-replicative complex assembly (6, 7) and stimulates phosphorylation of Mcm2-7 by Dbf4-Cdc7 kinase (8). Once Mcm10 is present, Cdc45 and GINS are loaded onto chromatin (6, 9, 10) and form a Cdc45/Mcm2-7/GINS helicase complex (11-14). Cyclinand Dbf4-dependent kinases together with Sld2, Sld3, and Dpb11 in budding yeast (15, 16) stimulate origin unwinding, which is signified by recruitment of replication protein A to singlestranded DNA (17, 18). Mcm10, Cdc45, and replication protein A facilitate subsequent loading of DNA polymerase ␣-primase (pol ␣) onto chromatin (7,9,19,20). The association of proliferating cell nuclear antigen, RFC, and replicative DNA polymerases ␦ and ⑀ with the origin completes the replisome (for review, see Ref. 21).A number of interactions have been observed between Mcm10 and proteins found in the pre-replicative complexes and at the replication fork. Mcm10 is a component of active replication complexes in Xenopus and budding yeast (12,14) and is associated with chromatin throughout S-phase (7). Mcm10 interacts genetically w...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 87%

Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

Robertson

Warren

Zhang

et al. 2008

Journal of Biological Chemistry

125

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“…Recent studies suggest that Mcm10 may function as a ring complex of six subunits (Cook et al 2003;Okorokov et al 2007). It is conceivable that individual subunits of the Mcm10 complex may interact with a different set of interactors.…”

Section: Discussionmentioning

confidence: 99%

Mcm10 Mediates the Interaction Between DNA Replication and Silencing Machineries

Tye

2009

Genetics

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The connection between DNA replication and heterochromatic silencing in yeast has been a topic of investigation for .20 years. While early studies showed that silencing requires passage through S phase and implicated several DNA replication factors in silencing, later works showed that silent chromatin could form without DNA replication. In this study we show that members of the replicative helicase (Mcm3 and Mcm7) play a role in silencing and physically interact with the essential silencing factor, Sir2, even in the absence of DNA replication. Another replication factor, Mcm10, mediates the interaction between these replication and silencing proteins via a short C-terminal domain. Mutations in this region of Mcm10 disrupt the interaction between Sir2 and several of the Mcm2-7 proteins. While such mutations caused silencing defects, they did not cause DNA replication defects or affect the association of Sir2 with chromatin. Our findings suggest that Mcm10 is required for the coupling of the replication and silencing machineries to silence chromatin in a context outside of DNA replication beyond the recruitment and spreading of Sir2 on chromatin.

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“…Biological responses elicited by mimosine may result from inhibition of zinc-dependent DNA binding metalloproteins including those involved in DNA replication initiation (25,26) and translation (27). Whereas the in vivo zinc chelating activity of mimosine and other iron chelators would be expected to be invariant among cell types, mimosine is a cell-specific inhibitor of both cell cycle and SHMT1 transcription.…”

Section: Identification Of Mimosine-responsive Transcriptionalmentioning

confidence: 99%

Mimosine Attenuates Serine Hydroxymethyltransferase Transcription by Chelating Zinc

Perry

Sastry

Nasrallah

et al. 2005

Journal of Biological Chemistry

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L-Mimosine is a naturally occurring plant amino acid and iron chelator that arrests the cell cycle in the late G 1 phase, although its mechanism of action is not known. Some studies indicate that mimosine prevents the initiation of DNA replication, whereas other studies indicate that mimosine disrupts elongation of the replication fork by impairing deoxyribonucleotide synthesis by inhibiting the activity of the iron-dependent enzyme ribonucleotide reductase and the transcription of the cytoplasmic serine hydroxymethyltransferase gene (SHMT1). In this study, the mechanism for mimosineinduced inhibition of SHMT1 transcription was elucidated. A mimosine-responsive transcriptional element was localized within the first 50 base pairs of the human SHMT1 promoter by deletion analyses and gel mobility shift assays. The 50-base-pair sequence contains a consensus zinc-sensing metal regulatory element (MRE) at position ؊44 to ؊38, and mutation of the MRE attenuated mimosine-induced transcription repression. Mimosine treatment eliminated MRE-and Sp1-binding activity in nuclear extracts from MCF-7 cells but not in nuclear extracts from a mimosine-resistant cell line, MCF-7/2a. MCF-7 cells cultured in zinc-depleted medium for more than 16 days were viable and lacked cytoplasmic serine hydroxymethyltransferase protein, confirming that mimosine inhibits SHMT1 transcription by chelating zinc. The disruption of DNA-protein interactions by zinc chelation provides a general mechanism for the inhibitory effects of mimosine on nuclear processes, including replication and transcription. Furthermore, this study establishes that SHMT1 is a zinc-inducible gene, which provides the first mechanism for the regulation of folate-mediated one-carbon metabolism by zinc.Mimosine is an amino acid found in plants of the Mimosa and Leucaena genera and a tyrosine analog (1). It is a cell-specific inhibitor of DNA replication in vivo; sensitive cell lines include human MCF-7 (2), HL60 (3) and Chinese hamster ovary (4), whereas human neuroblastoma (2), K562 (3), and Xenopus and mouse embryonic cells are resistant (5, 6). Mimosine is an effective iron chelator and a structural analog of deferiprone, a compound that is used clinically to lower systemic iron in patients who suffer from iron overload (7, 8) (Fig. 1).Mimosine arrests cell cycle progression in late G 1 and although it elicits a plethora of biological effects, its mechanism of action has not been established. Mimosine has been proposed to arrest DNA synthesis at the replication fork by repressing deoxyribonucleotide synthesis (9 -12). Mimosine and deferoxamine inhibit the activity of the iron-dependent enzyme ribonucleotide reductase in vitro by chelating iron (9, 11, 12) and deplete cellular deoxyribonucleotide pools in some but not all studies (2, 12). Furthermore, mimosine and deferoxamine have been shown to inhibit the transcription of the cytoplasmic serine hydroxymethyltransferase gene (SHMT1) 1 in MCF-7 cells but not in mimosine-resistant SH-SY5Y neuroblastoma (2). The cytoplasmic ser...

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A Novel Zinc Finger Is Required for Mcm10 Homocomplex Assembly

Cited by 36 publications

References 49 publications

Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

Mcm10 Mediates the Interaction Between DNA Replication and Silencing Machineries

Mimosine Attenuates Serine Hydroxymethyltransferase Transcription by Chelating Zinc

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