Interaction and Assembly of Murine Pre-replicative Complex Proteins in Yeast and Mouse Cells

Kneissl, Margot; Pütter, Vera; Szalay, Aladar A.; Grummt, Friedrich

doi:10.1016/s0022-2836(03)00079-2

Cited by 67 publications

(54 citation statements)

References 103 publications

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“…8). Associations between Orc2, Orc3, Orc4, and Orc5 proteins have been demonstrated in the cytoplasm of living cells, in the cytosol of cell extracts, in baculovirus expressed proteins, and in yeast two-hybrid screens (7,9,10,15). Orc2 plays a central role in maintaining ORC(2-5) within the nucleus, independent of its role in binding ORC to DNA.…”

Section: Nuclear Localization Of Orc6-humanmentioning

confidence: 99%

“…Human ORC assembled from baculovirus-expressed proteins either lacks Orc6 or it is present only in trace amounts (11)(12)(13)(14). Nevertheless, interactions between mammalian Orc6 and other ORC subunits have been detected by yeast two-hybrid screens (15), by association of ORC subunits in vitro (10,11), and by immunoprecipitation of ORC subunits from cell extracts (11). Thus, the interaction of mammalian Orc6 with other ORC subunits appears tenuous.…”

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confidence: 99%

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Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Ghosh

Vassilev

Zhang

et al. 2011

Journal of Biological Chemistry

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Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.

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Section: Nuclear Localization Of Orc6-humanmentioning

confidence: 99%

mentioning

confidence: 99%

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Ghosh

Vassilev

Zhang

et al. 2011

Journal of Biological Chemistry

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“…Genomic sequences upstream of the c-MYC gene were targeted through double ChIP using an antibody against Cdc6 for the first ChIP and followed by an anti-MCM8 antibody for the second ChIP. This procedure localized MCM8 with Cdc6, a protein reported to be involved in the subsequent loading of MCM proteins into the pre-RC (Kinoshita & Johnson, 2004;Kneissl et al, 2003;Lei & Tye, 2001). This localization was on specific DNA segments flanking the approximate center of the c-MYC replication initiation zone (Vassilev & Johnson, 1990), and occurred during both G1 and S phases, but not continuously.…”

Section: Research Regarding Mcm8 Protein Function In Dna Replication mentioning

confidence: 99%

Addressing the Enigma of MCM8 in DNA Replication

Daniel¹,

Johnson²

2011

Fundamental Aspects of DNA Replication

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“…Thus, Orc2 can neutralize the toxic effects of Orc1. Orc1 does not form a dimeric complex with Orc2 in vitro (Vashee et al, 2001), but does bind to the ORC(2-5) core complex (Dhar et al, 2001;Giordano-Coltart et al, 2005;Kneissl et al, 2003;Vashee et al, 2001). Therefore, suppression of Orc1 toxicity by co-expression with Orc2 would require that Orc2 stimulate synthesis of other ORC subunits.…”

Section: Induction Of Apoptosismentioning

confidence: 99%

“…In mammals, these changes appear to be specific for the largest ORC subunit, Orc1. The mammalian ORC consists of a stable core complex of ORC subunits Orc2, Orc3, Orc4 and Orc5 [referred to here as ORC (2)(3)(4)(5)] that associate weakly with Orc1 and Orc6 (Dhar et al, 2001;Giordano-Coltart et al, 2005;Kneissl et al, 2003;Vashee et al, 2001). Interaction of Orc1 with the ORC core complex in the presence of ATP is essential for binding ORC to DNA and for assembly of pre-RCs in vitro (GiordanoColtart et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis

Saha

Ghosh

Vassilev

et al. 2006

Journal of Cell Science

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Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell-cycle-dependent changes in its Orc1 subunit. Here, we show that Orc1 modifications such as mono-ubiquitylation and hyperphosphorylation that occur normally during S and G2-M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of pre-replication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induced p53-independent apoptosis, and Orc1 accumulated perinuclearly rather than uniformly throughout the nucleus. This behavior mimicked the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that did not induce apoptosis, prevented induction of apoptosis and restored uniform nuclear localization of Orc1. This would promote assembly of ORC-chromatin sites, such as occurs during the transition from M to G1 phase. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of `unmodified' Orc1.

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Interaction and Assembly of Murine Pre-replicative Complex Proteins in Yeast and Mouse Cells

Cited by 67 publications

References 103 publications

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Addressing the Enigma of MCM8 in DNA Replication

Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis

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