Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta.
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 M(r) 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.
Carastro
L Michael
LM
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA.
Tan
Cheng-Keat
CK
Selg
Manuel
M
Jack
Hans-Martin
HM
So
Antero G
AG
Downey
Kathleen M
KM
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002845:Chromatography / Q000379:methods; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D021122:Protein Subunits; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D021122:Protein Subunits; D012261:Ribonucleoproteins; D015534:Trans-Activators; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D020365:RNA Helicases; C101828:UPF1 protein, human