Black phosphorus quantum dots coordinated with a sulfonic ester of the titanium ligand are prepared and exhibit enhanced stability. In vitro and in vivo photoacoustic imaging applications demonstrate that the quantum dots can efficiently accumulate inside the tumor producing tumor profiles with high spatial resolution, demonstrating their potential as an efficient agent for photoacoustic imaging.

Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA remains open, especially the mechanism by which ATP is utilized for the task. Here we describe pre-steady state analysis of ATP hydrolysis, PCNA clamp opening and DNA binding by S. cerevisiae RFC, and present the first kinetic model of a eukaryotic clamp loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA, and bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC•ATP•PCNA (open) intermediate is ready for entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA•DNA release. This model enables quantitative understanding of the multi-step mechanism of a eukaryotic clamp loader, and furthermore facilitates comparative analysis of loaders from diverse organisms.

The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp·DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at ~2 s−1) and bind primer–template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC·ATP·PCNAopen·ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s−1). Dissociation of PCNA·ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.

Background: Crystal structures reveal direct clamp-DNA contacts whose functions are unclear. Results: Mutations of single N-terminal arginines/lysines in the inner PCNA ring affect DNA binding-linked steps in the clamp loading mechanism. Conclusion: Loss of individual PCNA-DNA contacts disrupts clamp loading and its interaction with DNA. Significance: DNA binding to specific locations within circular clamps influences other proteins that work with clamps on DNA.

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