title: Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana.
abstract: An open reading frame from Arabidopsis thaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of this Arabidopsis Twinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In this study, N-terminal truncations of ATH implicate residues 80-102 interact with the RNA polymerase domain (RPD). In addition, chimeric proteins, constructed using domains from ATH and the well-characterized T7 phage DNA primase-helicase gp4, were created to determine if the weak primase activity of ATH could be enhanced. Two chimeric proteins were constructed: ATHT7 contains the ZBD and RPD domains of ATH tethered to the helicase domain of T7, while T7ATH contains the ZBD and RPD domains of T7 tethered to the helicase domain of ATH. Both chimeric proteins were successfully expressed and purified in E. coli, and assayed for traditional primase and helicase activities. T7ATH was able to generate short oligoribonucleotide primers, but these primers could not be cooperatively extended by a DNA polymerase. Although T7ATH contains the ATH helicase domain, it exhibited few of the characteristics of a functional helicase. ATHT7 lacked primase activity altogether and also demonstrated only weak helicase activities. This work demonstrates the importance of interactions between structurally and functionally distinct domains, especially in recombinant, chimeric proteins.
authors.affiliation: Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA, 01610, USA, jamie.towle-weicksel@yale.edu.
authors.forename: Jamie B
authors.initials: JB
authors.lastname: Towle-Weicksel
authors.forename: Yun
authors.initials: Y
authors.lastname: Cao
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Crislip
authors.forename: David L
authors.initials: DL
authors.lastname: Thurlow
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Crampton
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019915:DNA Primase; D004265:DNA Helicases
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