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Significance Compaction of bulky DNA is a universal issue for all DNA-based life forms. Both chloroplasts and mitochondria maintain their own multicopy genomes organized as nucleoids, but the mechanism of DNA compaction remains obscure. Here, we discovered a chloroplast nucleoid protein (HBD1) that is highly similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p) in terms of possessing two DNA-binding high mobility group box (HMG-box) domains. Our analyses of HBD1 based on DNA origami/atomic force microscopy showed that HBD1 is capable of compacting DNA by introducing U-turns and cross-strand bridges with the two HMG-box domains, indicating that proteins with two HMG-box domains could compact DNA in both mitochondrial and chloroplast nucleoids., Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA–protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9–mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Resolution achieved Chloroplasts possess multiple copies of their own chloroplast DNA that are packaged into DNA-protein complexes known as nucleoids. The shape, number, and distribution of chloroplast nucleoids change markedly depending on the cell cycle, developmental stage, and nutritional environment. Kobayashi et al. identified Holliday junction resolvase as a key factor in the dynamism of chloroplast nucleoids in the unicellular green algae Chlamydomonas reinhardtii . The gene encoding the resolvase is ubiquitously conserved among green plants. Disruption or down-regulation of this gene also disturbed chloroplast nucleoid organization and segregation in the land plant Arabidopsis thaliana . Science , this issue p. 631