Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

doi:10.1073/pnas.0905281106

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

^aDepartment of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands;
^bWellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;
Departments of ^cPharmacology and
^fBiology, Medical School, University of Patras, 26500 Rio, Patras, Greece;
^dDepartment of Medical Oncology and Cancer Genomics Center, Laboratory of Experimental Oncology, University Medical Center Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands; and
^eEuropean Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany

↵¹Present address: Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
↵³Deceased July 27, 2008.
↵⁴Present address: Cancer Epigenetics and Biology Program (PEBC), Catalan Institute of Oncology, 08907 L'Hospitalet, Barcelona, Catalonia, Spain.

Edited by John Kuriyan, University of California, Berkeley, CA, and approved October 2, 2009
↵²P.J.G. and A.L. contributed equally to this work. (received for review May 14, 2009)

Abstract

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a “permissive” heterotrimer and an “inhibitory” heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

Footnotes

⁵To whom correspondence may be addressed. E-mail: a.perrakis{at}nki.nl or j.j.blow{at}dundee.ac.uk
Author contributions: V.D.M., R.H.M., J.J.B., S.T., and A.P. designed research; V.D.M., P.J.G., A.L., N.K., E.C., R.K., S.v.G., A.F., M.V.P., M.S.I., and D.I.S. performed research; Z.L., R.H.M., J.J.B., D.I.S., and S.T. contributed new reagents/analytic tools; V.D.M., P.J.G., Z.L., R.H.M., J.J.B., D.I.S., S.T., and A.P. analyzed data; and V.D.M. and A.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
The atomic coordinates and structure factors have been deposited in the Protein Data Bank. www.pdb.org (PDB ID code 2WVR).
This article contains supporting information online at www.pnas.org/cgi/content/full/0905281106/DCSupplemental.