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First published online 28 September 2004
doi: 10.1242/jcs.01405

Journal of Cell Science 117, 5221-5231 (2004)
Published by The Company of Biologists 2004
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Research Article

Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

Maria Rosa Lidonnici1, Rossella Rossi1, Sonia Paixão1, Ramiro Mendoza-Maldonado2, Roberta Paolinelli3, Caterina Arcangeli3, Mauro Giacca2,3, Giuseppe Biamonti1 and Alessandra Montecucco1,*

1 Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
2 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
3 NEST/INFM and Scuola Normale Superiore, Piazza dei Cavalieri 7, 56126 Pisa, Italy

* Author for correspondence (e-mail: montecucco{at}igm.cnr.it)

Accepted 8 July 2004

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.

Key words: ORC, Eukaryotic DNA replication, Heterochromatin, HP1, FRET


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