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Nucleic Acids Research, 2002, Vol. 30, No. 10 2232-2243
© 2002 Oxford University Press

Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta

L. Michael Carastro1, Cheng-Keat Tan2, Manuel Selg3, Hans-Martin Jack3, Antero G. So1,2 and Kathleen M. Downey1,2,*

1Department of Biochemistry and Molecular Biology and 2Department of Medicine, University of Miami School of Medicine, Miami, FL 33101, USA and 3Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger Center, University of Erlangen-Nurnberg, Erlangen, Germany

Delta helicase is a 5' to 3' DNA helicase that partially co-purifies with DNA polymerase delta (pol delta) from fetal bovine thymus tissue. We describe the resolution of delta helicase from pol delta on heparin–agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA–cellulose chromatography, unique-sequence RNA–agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent Mr of 115 kDa in SDS–PAGE, and photo-crosslinked to [{alpha}-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5' to 3' RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with delta helicase on western analysis, and delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol delta in vivo.

* To whom correspondence should be addressed at: Department of Medicine (R-99), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101, USA. Tel: +1 305 243 6304; Fax: +1 305 243 4519; Email: kdowney{at}med.miami.edu Present address:L. Michael Carastro, The Salk Institute for Biological Studies, Gene Expression Laboratories, La Jolla, CA 92037, USA


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