Abstract
Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS–PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.
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Acknowledgments
We thank Dr. Craig Thulin and Katie Southwick for assistance with the mass spectrometry and 2-D gel analysis, and Luis Alvarez, Johnathon Overson, Colton Kempton and Cindee Perry for assistance with some of the experiments. This work was supported by grants from the USDA and from the BYU Mentoring Environments Grant program.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s11103-011-9751-7
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Lassen, M.G., Kocchar, S. & Nielsen, B.L. Identification of a soybean chloroplast DNA replication origin-binding protein. Plant Mol Biol 76, 463–471 (2011). https://doi.org/10.1007/s11103-011-9736-6
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DOI: https://doi.org/10.1007/s11103-011-9736-6