Journal Article

Arabidopsis thaliana organelles mimic the T7 phage DNA replisome with specific interactions between Twinkle protein and DNA polymerases Pol1A and Pol1B

Miller, Justin, Peralta-Castro, Antolín, Nielsen, Brent L., Morley, Stewart A., Brieba, Luis G., Ridge, Perry G., Oliphant, Amanda, Ong, Kai Li, Aldous, Stephen

2019

Overview

Background Plant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana : a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis. Results Yeast-two-hybrid analysis reveals residues 120–295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins. Conclusions The interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Addition polymerization / Agriculture / Amino acids / Arabidopsis / Arabidopsis thaliana / Bacterial genetics / bacteriophages / Biochemistry and physiology / Biomedical and Life Sciences / Chloroplasts / Computational biology / Coupling / Cyanobacteria / Deoxyribonucleic acid / Direct-coupling-analysis / DNA / DNA helicase / DNA polymerase / DNA polymerases / DNA primase / DNA repair / DNA replication / DNA-directed DNA polymerase / DNA-directed RNA polymerase / domain / E coli / Genes / Genetic aspects / Genomes / Genomics / Homology / Life Sciences / Mammals / Mitochondria / Mitochondrial DNA / nuclear genome / Organellar DNA replication / Organelles / Peptides / Phages / Plant Sciences / Polymerase chain reaction / Primase / Proteins / Redundancy / Replication / Research Article / Residues / RNA / Thermophoresis / Tree Biology / Yeast / Yeast-two-hybrid / Zinc finger proteins