The Bloom's Syndrome Gene Product Interacts with Topoisomerase III

doi:10.1074/jbc.275.13.9636

The Bloom's Syndrome Gene Product Interacts with Topoisomerase III*

From the ^‡Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom,^§Rhône-Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, Vitry sur Seine Cedex, 94403 France, and the ^¶Department of Cellular Science, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

Abstract

Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIα, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIα proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIα. Furthermore, through characterizing a genetic interaction betweenBLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIα interaction domains in BLM.

Footnotes

↵* This work was supported by the Imperial Cancer Research Fund and Rhone-Poulenc Rorer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed. Tel.: 44-1865-222417; Fax: 44-1865-222431; E-mail: hickson@icrf.icnet.uk.
↵1 P. S. North et al., manuscript in preparation.
↵3 H. Turley et al., manuscript in preparation.
Abbreviations:

PCR

polymerase chain reaction

BSA

bovine serum albumin

DTT

dithiothreitol

kb

kilobase(s)

GST

glutathione S-transferase

MBP

maltose-binding protein

PBSA

phosphate buffered saline

PAGE

polyacrylamide gel electrophoresis

Pipes

1,4-piperazinediethanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

TBS

Tris-buffered saline
- Received September 23, 1999.
- Revision received December 8, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.