In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

Background:The ABCA4 protein is proposed to transport all-trans-retinal from the outer segment discs of retinal rod and cone photoreceptors. Results: 11-cis-Retinal bound specifically and with high affinity to the NBD1 domain of hABCA4. Conclusion: The NBD1 domain plays further roles in addition to nucleotide hydrolysis. Significance: ABCA4 may play a novel role in the visual transduction cycle involving 11-cis-retinal.

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 ؎ 3% ␣-helix, 20 ؎ 3% ␤-pleated sheet, and 53 ؎ 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal. ABC3 transporters are required for transport of a wide variety of hydrophobic substances across cellular membranes, including drugs (1-3), lipids (4 -6), metabolites, peptides (7), and steroids (2). Typically, eukaryotic ABC proteins are composed of two tandem sets of six transmembrane helices followed by a Walker type A and a type B nucleotide-binding motif (8). To date, 48 members of the human ABC transporter family have been identified, which, based on sequence homology, have been divided into seven subfamilies, ABCA through ABCG (2-3, 9).A retina-specific variant of the ABCA subfamily, the ABCA4 gene product (ABCR), was first described as the bovine and Xenopus Rim proteins identified in the rims of the rod outer segment discs (10 -11). Mutations in the ABCA4 gene appear to lead to defects in the energy-dependent transport of all-transretinal and lead to the accumulation of cytotoxic lipofuscin fluorophores in the retinal pigment epithelium characteristic of the diseases such as Stargardt disease, fundus flavimaculatus, and autosomal recessive cone-rod dystrophy, as well as increased susceptibility to age-related macular degeneration (12)(13)(14)(15)(16)(17)(18)(19)(20). Although ultimately these diseases await the promise of a cure through gene therapy (21-22), current treatments have been directed toward slowing the progression of the disease. Consequently, understanding how genetic mutations lead to retinal degeneration is critical for the development of novel therapies.Studies in several laboratories and the establishment of ABCA4 ...