Abstract
BackgroundThe Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts.ResultsTo determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis.ConclusionsThe results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.
Highlights
The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is homologous to the eukaryotic T7 gp4-like protein with intramitochondrial nucleoid localization (Twinkle) protein
Stronger intensity of the primers of 9 and 14 bases was consistently observed, and are similar in size to products reported for other DNA primases [22]
The primers were capable of being extended by DNA polymerase into high molecular weight DNA (Figure 2B), which is a fundamental property of a DNA primase that generates primers for DNA replication
Summary
The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp protein, which is homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. DNA replication involves the coordinated activity of several enzymes and proteins. These enzymes assist with the unwinding, separation, and copying of double stranded DNA to produce new identical DNA copies [1]. DNA helicase translocates unidirectionally along one strand of the nucleic acid to facilitate replication initiation. The helicase utilizes ATP hydrolysis to separate the DNA double helix into individual strands [2,3]. DNA polymerase uses the primers and extends the 3' end of the nucleotide chain by adding nucleotides matched to the template strand [1]
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