Identification of a soybean chloroplast DNA replication origin-binding protein.
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.
Department of Microbiology and Molecular Biology, Brigham Young University, 775 WIDB, Provo, UT 84602, USA.
Matthew G
MG
Lassen
Sunita
S
Kochhar
Sunita
S
Kocchar
Brent L
BL
Nielsen
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002736:Chloroplasts / Q000235:genetics*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000235:genetics*
D004268:DNA-Binding Proteins
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