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About 2,725 papers for “Origin recognition complex”

Characterisation of xenopus origin recognition complex.

Tamara Tugal · UCL Discovery (University College London) · 2000

The Origin Recognition Complex (ORC) of S. cerevisiae plays a crucial role in recognition of origins of DNA replication within the yeast genome. In Xenopus egg extracts DNA replication initiates from random DNA sequences suggesting that origins of DNA replication are not required. Despite this apparent lack of requirement for DNA sequences serving as origins of DNA replication, a protein homologous to the Ore Ip subunit of yeast ORC has been identified in Xenopus. This discovery has indicated that the mechanism which recognises replication origins in higher eukaryotes could be similar to that in yeast. Work described in this thesis started at the time when Xenopus Ore Ip was identified. In the quest to understand Xenopus ORC and it's function in origin recognition I set out to purify this protein complex.
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\nI produced monoclonal antibodies which recognised Xenopus Ore Ip subunit of ORC with high affinity and specificity. Using these antibodies, I purified a protein complex which contained Xenopus Ore Ip in association with at least four other polypeptides. I showed that one of these polypeptides was Xenopus Orc2p which was meanwhile identified by an independent research. Microsequencing of another Ore Ip associated protein led to identification of mouse Orc4 gene from the EST database. I used the identified mouse Orc4 sequence to screen Xenopus cDNA library and I cloned Xenopus Orc4 gene. Microsequencing of another two proteins co-purified with Ore Ip confirmed that they were Xenopus Orc3p and Orc5p and helped identification of their corresponding genes from frogs and humans.
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\nI described a mobility shift of Xenopus Ore Ip occurring upon the exit from mitosis and showed that it correlated with dephosphorylation of the protein. I determined that the amount of ORC present in a single frog egg is 100 000 fold higher than in a single somatic cell.
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\nFinally, I showed that ORC purified from Xenopus egg extract binds to S. cerevisiae ARSl sequence, as well as to DNA sequences derived from a bacterial plasmid. I observed that this ORC-DNA interaction was dependent on ATP. However, the way by which ORC determines the sites of initiation of DNA replication in higher eukaryotes remains unknown.

Cited by 0

REVIEW The origin recognition complex: from

Stephen P. Bell · 2013

Eukaryotic DNA replication is an intricate process that requires the coordinate and tightly regulated action of numerous molecular machines. Eukaryotic cells must choose hundreds if not thousands of origins of replication and assemble multiple proteins or protein complexes at these sites. During S phase, these complexes are activated in a reproducible, temporal program to generate twice this number of replication fork machines dedicated to DNA synthesis. Moreover, these machines must overcome the highly packaged nature of the eukaryotic genome to access all DNA sites, without exception. The key factor in the selection of initiation sites and the assembly of the replication machinery is the initiator protein. Originally proposed by Brenner, Cuzin, and Jacob based on genetic findings, the replicon model hypothesized

Cited by 0

Humanizing the yeast origin recognition complex

Clare S K Lee, Ming Fung Cheung, Jinsen Li et al. · Nature Communications · 2021

The Origin Recognition Complex (ORC) is an evolutionarily conserved six-subunit protein complex that binds specific sites at many locations to coordinately replicate the entire eukaryote genome. Though highly conserved in structure, ORC's selectivity for replication origins has diverged tremendously between yeasts and humans to adapt to vastly different life cycles. In this work, we demonstrate that the selectivity determinant of ORC for DNA binding lies in a 19-amino acid insertion helix in the Orc4 subunit, which is present in yeast but absent in human. Removal of this motif from Orc4 transforms the yeast ORC, which selects origins based on base-specific binding at defined locations, into one whose selectivity is dictated by chromatin landscape and afforded with plasticity, as reported for human. Notably, the altered yeast ORC has acquired an affinity for regions near transcriptional start sites (TSSs), which the human ORC also favors.

Cited by 53Open access

Nonreplicative functions of the origin recognition complex

V. V. Popova, Alexander Brechalov, С. Г. Георгиева et al. · Nucleus · 2018

Origin recognition complex (ORC), a heteromeric six-subunit complex, is the central component of the eukaryotic pre-replication complex. Recent data from yeast, frogs, flies and mammals present compelling evidence that ORC and its individual subunits have nonreplicative functions as well. The majority of these functions, such as heterochromatin formation, chromosome condensation, and segregation are dependent on ORC-DNA interactions. Furthermore, ORC is involved in the control of cell division via its participation in centrosome duplication and cytokinesis. Recent findings have also demonstrated a direct interaction between ORC and mRNPs and highlighted an essential role of ORC in mRNA nuclear export. Along with the growth of evolutionary complexity of organisms, ORC complex functions become more elaborate and new functions of the ORC sub-complexes and individual subunits have emerged.

Cited by 28Open access

Architecture of the Human Origin Recognition Complex

Suman Kumar Dhar, Laurie M. Delmolino, Anindya Dutta · Journal of Biological Chemistry · 2001

All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far. However, not much has been reported on the nature and the characteristics of the human origin recognition complex. In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4, and -5 form a core complex. HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2-5 complex is extremely labile. We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3. The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex. A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction. Overexpression of this fragment in U2OS cells blocked the cells in G(1), providing the first evidence that a mammalian ORC subunit is important for the G(1)-S transition in mammalian cells.

Cited by 127Open access

Assembly of the Human Origin Recognition Complex

Sanjay Vashee, Pamela Simancek, M D Challberg et al. · Journal of Biological Chemistry · 2001

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.

Cited by 158Open access

The Origin Recognition Complex Marks a Replication Origin in the

Human Top, Gene Promoter, Christian Keller et al. · 2002

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin im-munoprecipitation. Antibodies specific for hOrc2 pro-tein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island up-stream of the human TOP1 gene. Nascent strand abun-dance assays show that the ORC binding region coin-cides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix at-tachment regions. The matrix attachment region ele-ments analyzed contain no ORC and constitute no sites

Cited by 0

The origin recognition complex: from simple origins to complex functions

Stephen P. Bell · Genes & Development · 2002

Eukaryotic DNA replication is an intricate process that requires the coordinate and tightly regulated action of numerous molecular machines. Eukaryotic cells must choose hundreds if not thousands of origins of replication and assemble multiple proteins or protein complexes at these sites. During S phase, these complexes are activated in a reproducible, temporal program to generate twice this number of replication fork machines dedicated to DNA synthesis. Moreover, these machines must overcome the highly packaged nature of the eukaryotic genome to access all DNA sites, without excep

Cited by 286Open access