Identification and characterization of DCP68 as a bifunctional DNA-binding nucleoid protein and sulfite reductase
The biochemical characterization of the D[barbelow]NA c[barbelow]ompacting p[barbelow]rotein of 6[barbelow]8[barbelow] kDa from chloroplasts of soybean SB-M cells was continued for the purpose of understanding the role nucleoid proteins may play in determining genome structure and function. Previously, DCP68 has been shown to associate with DNA by forming nucleoid-like structures and inhibit DNA replication in vitro (38). In this study, amino acid sequence and spectrophotometric analyses as well as immunological crossreactivity with antibodies raised against Arabidopsis thaliana sulfite reductase identified DCP68 as ferredoxin:sulfite reductase, a siroheme enzyme involved in sulfur assimilation. The association of DCP68 with chloroplast nucleoids was confirmed using in organello formaldehyde crosslinking of the protein-DNA complexes and co-immunocolocalization by fluorescence microscopy. The phosphorylation state of DCP68 was shown to affect its interactions with DNA in vitro , indicating that this may be a way of regulating its activity in vivo . Finally, a recombinant Arabidopsis sulfite reductase protein was expressed in E. coli and used to produce antibodies that recognize DCP68/SiR. Hypotheses for the physiological significance of DCP68's bifunctionality as a protein that participates in plastid DNA organization and sulfur assimilation were discussed.