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Molecular and Cellular Biology, January 2005, p. 172-184, Vol. 25, No. 1
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.1.172-184.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Composition and Architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) Complex

John Sergeant,1,{dagger} Elaine Taylor,1,{dagger} Jan Palecek,1,{dagger} Maria Fousteri,1,{ddagger} Emily A. Andrews,1 Sara Sweeney,1 Hideo Shinagawa,2 Felicity Z. Watts,1 and Alan R. Lehmann1*

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, United Kingdom,1 Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan2

Received 21 May 2004/ Returned for modification 30 June 2004/ Accepted 24 September 2004

The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes. Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex. We show here that both S. pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region. This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5). We purified the Smc5-6 complex from S. pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62. Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue. In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62. The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3.

* Corresponding author. Mailing address: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom. Phone: 44-1273 678120. Fax: 44-1273 678120. E-mail: a.r.lehmann{at}sussex.ac.uk.

{dagger} J.S., E.T., and J.P. contributed equally to this work.

{ddagger} Present address: Department of Toxicogenetics, Leiden University Medical Centre, 2333-AL Leiden, The Netherlands.

Molecular and Cellular Biology, January 2005, p. 172-184, Vol. 25, No. 1
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.1.172-184.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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