Title

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

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Kathleen M. Downey, Committee Chair

Second Committee Member

Antero G. So, Committee Member

Abstract

DNA polymerase delta (pol delta), the primary replicative DNA polymerase in eukaryotes, partially co-purifies with a 5' to 3' DNA helicase, delta helicase, from calf thymus tissue, but the two enzymes can be separated by chromatography on heparin-agarose. Following heparin-agarose chromatography, delta helicase was purified to apparent homogeneity by chromatography on single-stranded DNA-cellulose, a unique sequence RNA-cellulose and ceramic hydroxyapatite. Delta helicase isolated from calf thymus had an Mr of 115-kDa on SDS-PAGE, and photocrosslinked to [alpha- 32P]ATP. Microcapillary reverse-phase HPLC nano-elecotrospray tandem mass spectrometry peptide sequence analysis indicated that the amino acid sequences of the tryptic peptides derived from delta helicase matched the protein sequence of HUPF1, a 5' to 3' RNA helicase which can unwind both DNA and RNA substrates in vitro. HUPF1 is a requisite component of the nonsense-mediated mRNA decay process (NMD) whereby mRNAs containing premature termination codons are selectively degraded. Antisera against HUPF1 crossreacted with delta helicase on Western analysis, and delta helicase activity was immunoinactivated by preincubation with antibodies to HUPF1. The biochemical properties of delta helicase, HUPF1 and yeast Upf1 were found to be essentially identical, suggesting that delta helicase is the bovine homologue of HUPF1. A physical interaction between HUPF1 and the 66-kDa third subunit of pol delta was demonstrated by co-immunoprecipitation of the two proteins from HeLa cells transiently transfected with plasmids encoding either epitope-tagged HUPF1 or epitope-tagged p66, suggesting that mammalian Upf1 and pol delta may interact in vivo.

Keywords

Biology, Molecular; Biology, Cell; Biogeochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3050714