Anti-Angiogenic and Anti-Scarring Dual Action of an Anti-Fibroblast Growth Factor 2 Aptamer in Animal Models of Retinal Disease.

Currently approved therapies for age-related macular degeneration (AMD) are inhibitors against vascular endothelial growth factor (VEGF), which is a major contributor to the pathogenesis of neovascular AMD (nAMD). Intravitreal injections of anti-VEGF drugs have shown dramatic visual benefits for AMD patients. However, a significant portion of AMD patients exhibit an incomplete response to therapy and, over the extended management course, can lose vision, with the formation of submacular fibrosis as one risk factor. We investigated a novel target for AMD treatments, fibroblast growth factor 2 (FGF2), which has been implicated in the pathophysiology of both angiogenesis and fibrosis in a variety of tissue and organ systems. The anti-FGF2 aptamer, RBM-007, was examined for treatment of nAMD in animal models. In in vivo studies conducted in mice and rats, RBM-007 was able to inhibit FGF2-induced angiogenesis, laser-induced choroidal neovascularization (CNV), and CNV with fibrosis. Pharmacokinetic studies of RBM-007 in the rabbit vitreous revealed high and relatively long-lasting profiles that are superior to other approved anti-VEGF drugs. The anti-angiogenic and anti-scarring dual action of RBM-007 holds promise as an additive or alternative therapy to anti-VEGF treatments for nAMD.

A replicase clamp-binding dynamin-like protein promotes colocalization of nascent DNA strands and equipartitioning of chromosomes in E. coli.

In Escherichia coli, bidirectional chromosomal replication is accompanied by the colocalization of sister replication forks. However, the biological significance of this mechanism and the key factors involved are still largely unknown. In this study, we found that a protein, termed CrfC, helps sustain the colocalization of nascent DNA regions of sister replisomes and promote chromosome equipartitioning. CrfC formed homomultimers that bound to multiple molecules of the clamp, a replisome subunit that encircles DNA, and colocalized with nascent DNA regions in a clamp-binding-dependent manner in living cells. CrfC is a dynamin homolog; however, it lacks the typical membrane-binding moiety and instead possesses a clamp-binding motif. Given that clamps remain bound to DNA after Okazaki fragment synthesis, we suggest that CrfC sustains the colocalization of sister replication forks in a unique manner by linking together the clamp-loaded nascent DNA strands, thereby laying the basis for subsequent chromosome equipartitioning.

Structure and function of DnaA N-terminal domains: specific sites and mechanisms in inter-DnaA interaction and in DnaB helicase loading on oriC.

DnaA forms a homomultimeric complex with the origin of chromosomal replication (oriC) to unwind duplex DNA. The interaction of the DnaA N terminus with the DnaB helicase is crucial for the loading of DnaB onto the unwound region. Here, we determined the DnaA N terminus structure using NMR. This region (residues 1-108) consists of a rigid region (domain I) and a flexible region (domain II). Domain I has an alpha-alpha-beta-beta-alpha-beta motif, similar to that of the K homology (KH) domain, and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggest a model for DnaA homomultimer formation and DnaB helicase loading on oriC.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding.