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

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          Abstract

          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.

          Electronic supplementary material

          The online version of this article (10.1186/s12870-019-1854-3) contains supplementary material, which is available to authorized users.

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          Most cited references 55

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          Analyzing real-time PCR data by the comparative CT method

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            UniProt: the universal protein knowledgebase

            Nucleic Acids Research (2017) 45: D158–D169, https://doi.org/10.1093/nar/gkw1099 The authors wish to make the following correction to their article. Co-author Alexandre Renaux affiliated with the EMBL-EBI was mistakenly omitted from the list of authors. His name has been added to the full list of authors in the Acknowledgement section of the published article.
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              Predicting Secretory Proteins with SignalP.

              SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.
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                Author and article information

                Contributors
                stewmorley@gmail.com
                antolin.peralta@cinvestav.mx
                luis.brieba@cinvestav.mx
                justin@ke7gts.com
                jkm525@go.byuh.edu
                perry.ridge@byu.edu
                aeoliphant@gmail.com
                stephen.r.aldous@gmail.com
                1-801-422-1102 , brentnielsen@byu.edu
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                6 June 2019
                6 June 2019
                2019
                : 19
                Affiliations
                [1 ]ISNI 0000 0004 1936 9115, GRID grid.253294.b, Department of Microbiology & Molecular Biology, , Brigham Young University, ; 3130 Life Sciences Building, 4007 LSB, Provo, UT 84604 USA
                [2 ]Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, 36821 Irapuato, Guanajuato Mexico
                [3 ]ISNI 0000 0004 1936 9115, GRID grid.253294.b, Department of Biology, , Brigham Young University, ; 4007 LSB, Provo, UT 84604 USA
                Article
                1854
                10.1186/s12870-019-1854-3
                6554949
                31170927
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: CONACYT
                Award ID: 253757
                Funded by: FundRef http://dx.doi.org/10.13039/100006757, Office of Research and Creative Activities, Brigham Young University;
                Award ID: 2005
                Funded by: FundRef http://dx.doi.org/10.13039/100006756, Brigham Young University;
                Award ID: 2016
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

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