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

Abstract

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.

Figure 1

Elution profiles of pol delta and delta helicase activities on heparin–agarose chromatography. DNA polymerase activity was assayed using poly(dA)/oligo(dT) as template/primer in the presence (closed squares) and absence (open squares) of PCNA, as described in Materials and Methods, and is reported as pmol dTMP incorporated/15 min. Helicase activity was assayed in the presence (closed circles) and absence (open circles) of ATP using a fork-like substrate constructed from single-stranded M13 (+) DNA and a partially annealed 5′-[³²P]-50mer, as described in Materials and Methods, and is reported as integrated volume of displaced 50mer. The KCl concentration of the fractions (open triangles) is also indicated (mM).

Figure 2

Affinity purification of delta helicase on FPLC ssDNAc chromatography. Helicase activity was assayed in the presence (A) and absence (B) of ATP as described in Materials and Methods. Depicted in (A) and (B) are phosphorimages of helicase assays resolved on non-denaturing 8% PAGE gels. The migration positions of the fork-like helicase substrate (Substrate) and the displaced 50mer (Product) are indicated to the left. The letters above the lanes are: H, substrate heated at 90°C for 10 min as a positive control; U, unheated substrate as a negative control; BC, before column sample; FT, flow through sample after loading. The numbers above the lanes indicate the column fraction numbers. (C) Aliquots of the ssDNAc column fractions were resolved by SDS–PAGE and polypeptide bands were detected by silver staining, as described in Materials and Methods. (D) Aliquots of the ssDNAc column fractions were resolved by SDS–PAGE and western analysis using a monoclonal antibody against the human hnRNP L protein were performed as described in Materials and Methods. For (C and D), the letters above the lanes are: BC, before column sample; M, molecular weight markers with the known kDa position indicated to the left. The numbers above the lanes indicate the column fraction numbers. For (A)–(D), fractions 59–71 eluted with 1 M KCl, and fractions 87–97 eluted with 2 M KCl.

Figure 3

Resolution of delta helicase and hnRNP L on FPLC TK119/RNA–Sepharose chromatography. (A) Helicase activity was assayed in the presence of ATP, reaction products were resolved on 8% non-denaturing PAGE gels, and products were detected by phosphorimaging as described in Materials and Methods. The migration positions of the helicase substrate (S) and the displaced 50mer reaction product (P) are indicated to the left. The letters above the lanes are: BC, before column sample; FT, flow through sample after loading. The numbers above the lanes indicate the fraction numbers. (B) Aliquots of the TK119/RNA–Sepharose column fractions were resolved by SDS–PAGE and polypeptide bands were detected by silver staining, as described in Materials and Methods. (C) Aliquots of the TK119/RNA–Sepharose column fractions were resolved by 10% SDS–PAGE gels and western analysis was carried out as described in Materials and Methods, using a monoclonal antibody against the human hnRNP L protein. For (B) and (C), the letters above the lanes are: BC, before column sample; FT, flow through sample after loading; M, molecular weight markers with the known kDa position indicated to the left. The numbers above the lanes indicate the fraction numbers. For (A)–(C), fractions 4–20 eluted with 75 mM KCl, and fractions 24–40 eluted with 150 mM KCl.

Figure 4

Purification of delta helicase on CHA FPLC chromatography. (A) Helicase activity was assayed in the presence of ATP and reaction products were resolved on 8% non-denaturing PAGE gels and detected by phosphorimaging as described in Materials and Methods. The migration positions of the fork-like helicase substrate (Substrate) and the displaced 50mer reaction product (Product) are indicated to the left. The letters above the lanes are: H, substrate heated at 90°C for 10 min as a positive control; U, unheated substrate as a negative control; BC, before column sample; F1 and F2, flow through samples after loading. The numbers above the lanes indicate the fraction numbers. (B) Aliquots of the column fractions were resolved by 10% SDS–PAGE gels and polypeptide bands were detected by silver staining, as described in Materials and Methods. The numbers above the lanes indicate the fraction numbers. M, molecular weight markers with the known kDa position indicated to the left.

Figure 5

Purified delta helicase UV photo-crosslinks with [α-³²P]ATP. Purified delta helicase (Fig. 4, fraction 55) was concentrated ∼50-fold and samples were photo affinity-labeled with [α-³²P]ATP by irradiating with UV, as described in Materials and Methods. (A) Photo-crosslinked reaction products were resolved on 10% SDS–PAGE gels and [³²P]-labeled products were visualized by autoradiography. (B) Polypeptides were visualized by staining with a Colloidal Blue Staining Kit. The arrows indicate the positions of the 112- and 115-kDa polypeptides. For (A) and (B), the labels above the lanes are: M, molecular weight markers with the known kDa positions indicated to the left; Helicase, concentrated homogeneous delta helicase sample (∼0.4 µg); BSA, bovine serum albumin (3 µg).

Figure 6

Tandem mass spectrometry data from delta helicase and sequence of HUPF1. The complete 1129 amino acid sequence deduced from the human HUPF1 cDNA is given. Tryptic peptides of the purified 115-kDa delta helicase polypeptide were subjected to µLC/MS/MS analysis. The tryptic peptide mass data derived from delta helicase matched with the predicted masses for the HUPF1 regions that are indicated in red letters. Two unique zinc finger motifs (ZnFr1 and ZnFr2) and the seven classic helicase domains (Ia, Ib, II, III, IV, V, VI) are underlined. Putative zinc finger motif cysteine (C) and histidine (H) residues are in a larger font size. Residues within the putative zinc finger motifs that are conserved in mammalian, C.elegans, S.cerevisiae and S.pombe Upf1 homologs are indicated by blue letters, and those conserved residues that overlap with the delta helicase sequence are indicated by purple letters. Residues conserved in the Superfamily I helicase domains are in bold.

Figure 7

Immunoinactivation of delta helicase. Immunoinactivation reactions were performed and quantified as described in Materials and Methods and the results represented in the histogram. Briefly, helicase reactions were preincubated with one of four antibodies tested, in three different quantities (either 5.5, 11 or 22 µg of IgG). Reaction products were resolved on 8% PAGE, then detected by phosphorimaging and quantified with ImageQuant (Molecular Dynamics). The results were entered in an Excel (Microsoft) spreadsheet, the percent of the total substrate consumed was calculated, and then converted into a histogram. The abbreviations under the histogram columns are: PreImm, pre-immune antibodies; HUPF1, antipeptide antibody against HUPF1; p125, antibody against an N-terminal pol delta p125 subunit–GST fusion protein; p66, antipeptide antibody against the pol delta p66 subunit.

Figure 8

Co-immunoprecipitation of pol delta p66 and HUPF1. Immunoprecipitation complexes were collected from HeLa cells transfected with one of five pCMV-cMyc expression vectors. The complexes were resolved on 10% SDS–PAGE and western analyses were carried out, followed as described in Materials and Methods. (A) Western analysis for endogenous p66 with antipeptide antiserum against the pol delta p66 subunit. (B) Western analysis for endogenous HUPF1 with affinity purified antipeptide antibodies against HUPF1. (C) Western analysis for cMyc with a monoclonal antibody against cMyc (9E10) conjugated to HRP. For (A)–(C) the abbreviations to the right of TX: and above the lanes indicate the pCMV-cMyc expression construct that the HeLa cells were transfected with, as described in Materials and Methods: HUPF1, pCMV-Myc/HUPF1; p66, pCMV-Myc/p66; p50, pCMV-Myc/p50; lacZ, pCMV-Myc/β-galactosidase; bla, pCMV-Myc/β-lactamase. The known positions of p66, HUPF1 and molecular weight markers are indicated to the left. (D) The transfected HeLa cell lysates were resolved by 10% SDS–PAGE and cMyc tagged proteins were detected by western analyses with a monoclonal antibody against cMyc (9E10) conjugated to HRP, followed by fluorography, as described in Materials and Methods. The detected bands were quantified with ImageJ (NCBI) and the measured mean gray values are depicted in an Excel (Microsoft) histogram. The transfected HeLa cell lysates are indicated below the columns, and the mean gray values are indicated on the vertical axis.

Figure 9

Western analyses of proteins resolved on heparin–agarose chromatography. Aliquots of the heparin–agarose fractions depicted in Figure 1 were subjected to 10% SDS–PAGE, followed by western analyses using (A) antipeptide antibodies against HUPF1, (B) antibodies against pol delta p125, (C) a monoclonal antibody against hnRNP L, (D) antipeptide antibodies against p66, and (E) antiserum against p50. For (A)–(E), the letters above the lanes are: BC, before column sample; L, flow through sample after loading; W, wash sample. The numbers above the lanes indicate the fraction number. The known positions of the molecular weight markers are indicated to the left.