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Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

ABSTRACT: In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.

SUBMITTER: McNairn AJ

PROVIDER: S-EPMC1350721 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

McNairn Adrian J AJ Okuno Yukiko Y Misteli Tom T Gilbert David M DM

Experimental cell research 20050801 2

In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin ...[more]

PMID: 15950218

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Project description:Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.

Dataset Information

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

Publications

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

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