Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

pH-Responsive Poly(ethylene glycol)-b-poly(2-vinylpyridine) Micelles for the Triggered Release of Therapeutics.

The use of pH-responsive polymeric micelles is a promising approach to afford the targeted, pH-mediated delivery of hydrophobic drugs within the low-pH tumour milieu and intracellular organelles of cancer cells. However, even for a common pH-responsive polymeric micelle system-e.g., those utilising poly(ethylene glycol)-b-poly(2-vinylpyridine) (PEG-b-PVP) diblock copolymers-there is a lack of available data describing the compatibility of hydrophobic drugs, as well as the relationships between copolymer microstructure and drug compatibility. Furthermore, synthesis of the constituent pH-responsive copolymers generally requires complex temperature control or degassing procedures that limit their accessibility. Herein we report the facile synthesis of a series of diblock copolymers via visible-light-mediated photocontrolled reversible addition-fragmentation chain-transfer polymerisation, with a constant PEG block length (90 repeat units (RUs)) and varying PVP block lengths (46-235 RUs). All copolymers exhibited narrow dispersity values (D ≤ 1.23) and formed polymeric micelles with low polydispersity index (PDI) values (typically <0.20) at physiological pH (7.4), within a suitable size range for passive tumour targeting (<130 nm). The encapsulation and release of three hydrophobic drugs (cyclin-dependent kinase inhibitor (CDKI)-73, gossypol, and doxorubicin) were investigated in vitro at pH 7.4-4.5 to simulate drug release within the tumour milieu and cancer cell endosome. Marked differences in drug encapsulation and release were observed when the PVP block length was increased from 86 to 235 RUs. With a PVP block length of 235 RUs, the micelles exhibited differing encapsulation and release properties for each drug. Minimal release was observed for doxorubicin (10%, pH 4.5) and CDKI-73 exhibited moderate release (77%, pH 4.5), whereas gossypol exhibited the best combination of encapsulation efficiency (83%) and release (91% pH 4.5) overall. These data demonstrate the drug selectivity of the PVP core, where both the block molecular weight and hydrophobicity of the core (and accordingly the hydrophobicity of the drug) have a significant effect on drug encapsulation and release. These systems remain a promising means of achieving targeted, pH-responsive drug delivery-albeit for select, compatible hydrophobic drugs-which warrants their further investigation to develop and evaluate clinically relevant micelle systems.