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.

hUPF2 Silencing Identifies Physiologic Substrates of Mammalian Nonsense-Mediated mRNA Decay

“…For example, yeast Upf1p is involved in translational termination (67), and hUPF1 is required for skipping of PTC-containing exons in human cells (46). Further, hUPF1 could modulate replication/transcription processes since it copurifies with DNA polymerase delta (10). Finally, hUPF1 could play additional roles in splicing, intracellular transport, polarization, and stabilization of certain mRNAs.…”

Telomeres: The silence is broken

“…Human UPF1 is a 5'-3' DNA and RNA helicase with nucleic acid-dependent ATPase and ATP-dependent activities. 40,41 The C-terminal PIN domain of hEST1A exhibits an overall structure similar to ribonucleases of the RNase H family and displays single stranded RNA endonuclease activity under certain experimental conditions. 42 UPF1 and hEST1A mutants lacking specific enzymatic activities will be used to understand if UPF1-mediated unwinding and hEST1A-mediated cleavage of TERRA molecules play a role in TERRA displacement from chromatin.…”

“…31 Interestingly, human UPF1 physically interacts with DNA polymerase δ, accumulates on chromatin during S-phase and is essential to complete DNA replication. 36,41 We speculate that UPF1 (as well as other SMG factors) might ensure proper telomere replication by displacing TERRA from telomeres during progression of the replication fork, thereby avoiding unwanted annealing of TERRA to single stranded telomeric DNA. Lack of such a displacement mechanism could lead to arrest of the replication fork and ultimately generate double stranded breaks in the telomeric tract.…”

The Double Life of UPF1 in RNA and DNA Stability Pathways

“…6 Indeed, UPF1 protein prepared form calf thymus copurifies extensively with polδ; 18 in addition, human UPF1 coimmunoprecipitates with the p66 subunit of polδ and the p125 catalytic subunit of polδ in HeLa nuclear extracts. 5,18 Importantly, while UPF1 also binds to UPF2, UPF2 does not coimmunoprecipitate with p125. 5 We therefore propose that UPF1 forms different complexes in vivo: one complex, containing UPF2, is responsible for NMD and a second one, comprising the polδ holoenzyme, is involved in DNA metabolism.…”

“…UPF1 is a 5' to 3' DNA and RNA helicase with nucleic acid-dependent ATPase activity. [15][16][17][18] A straightforward hypothesis is that chromatin-bound UPF1 works as a replicative helicase unwinding the DNA in front of the replication fork (Fig. 1B).…”