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.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence

“…Both recombinant proteins were purified to homogeneity after three chromatographic steps, with similar purity as a recombinant primase module (residues 1–262) from the T7 primase-helicase gene used as a control during this study (Figure 2A ). Previous biochemical characterizations of AtTwinkle indicated that this protein is active on single-stranded M13 templates ( 28 , 29 , 32 ). To assess if our purified proteins exhibit oligoribonucleotide synthesis activity we used a random heptaoligonucleotide as a template and assayed AtPrimase and T7 primase fragments for ribonucleotide synthesis.…”

“…Motif I corresponds to the ZBD necessary for template recognition ( 26 ), whereas regions II to VI are located in the RNAP domain. In vitro characterization of AtTwinkle demonstrated that this enzyme has both primase and helicase activities ( 28 , 29 , 32 ). However, its preferred ssDNA template sequence, its putative role in organellar replication, and its interactions with plant organellar DNA polymerases remain unclear.…”

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain

“…It is proposed that the ZFD fulfills roles in template recognition, primer stability, and primer transfer [ 5 , 18 , 20 , 21 ]. The Twinkle ortholog of the plant model Arabidopsis thaliana (AtTwinkle) is active, both as a primase and as a helicase [ 13 , 22 , 23 ]. Contrary to all primases characterized to date, the primase module of AtTwinkle recognizes two nucleotides (instead of one) as cryptic elements.…”