Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy

Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule

Kopilevich S, Müller A, Weinstock IA. Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule. Journal of the American Chemical Society. 2015;137(40):12740-12743.Using the hydrolysis of epoxides in water as a model reaction, the effect of multiple active sites on Michaelis-Menten compliant rate accelerations in a porous capsule is demonstrated. The capsule is a water-soluble 4-symmetry Keplerate-type complex of the form, [{(Mo6O21)-O-VI(H2O)(612){(Mo2O4)-O-V(L)}(30)](42-), in which 12 pentagonal "ligands," {(Mo-VI)(Mo5O2)-O-VI, (H2O)(6)}(6-), are coordinated to 30 dimolybdenum sites, {(Mo2O4L)-O-V}(1+) (L = an endohedrally coordinated 772-bound carboxylate anion), resulting in 20 MO9O9 pores. When "up-regulated" by removal of ca. one-third of the blocking ligands, L, an equal number of dimolybdenum sites are activated, and the newly freed-up space allows for encapsulation of nearly twice as many substrate guests, leading to a larger effective molarity (amplification), and an increase in the rate acceleration (k(cat)/L-uncat) from 16,000 to an enzyme-like value of 182,800

Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule

Using the hydrolysis of epoxides in water as a model reaction, the effect of multiple active sites on Michaelis–Menten compliant rate accelerations in a porous capsule is demonstrated. The capsule is a water-soluble <i>I</i>_<i>h</i>-symmetry Keplerate-type complex of the form, [{Mo^VI₆O₂₁(H₂O)₆}₁₂{Mo^V₂O₄(L)}₃₀]^42–, in which 12 pentagonal “ligands,” {(Mo^VI)­Mo^VI₅O₂₁(H₂O)₆}^6–, are coordinated to 30 dimolybdenum sites, {Mo^V₂O₄L}¹⁺ (L = an endohedrally coordinated η²-bound carboxylate anion), resulting in 20 Mo₉O₉ pores. When “up-regulated” by removal of ca. one-third of the blocking ligands, L, an equal number of dimolybdenum sites are activated, and the newly freed-up space allows for encapsulation of nearly twice as many substrate guests, leading to a larger effective molarity (amplification), and an increase in the rate acceleration (<i>k</i>_cat/<i>k</i>_uncat) from 16,000 to an enzyme-like value of 182,800

The Uptake and Assembly of Alkanes within a Porous Nanocapsule in Water: New Information about Hydrophobic Confinement

Kopilevich S, Gottlieb H, Keinan-Adamsky K, Müller A, Weinstock IA. The Uptake and Assembly of Alkanes within a Porous Nanocapsule in Water: New Information about Hydrophobic Confinement. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2016;55(14):4476-4481.In Nature, enzymes provide hydrophobic cavities and channels for sequestering small alkanes or long-chain alkyl groups from water. Similarly, the porous metal oxide capsule [{(Mo6O21)-O-VI(H2O)(6)}(12){((Mo2O4)-O-V)(30)(L)(29)(H2O)(2)}](41-) (L=propionate ligand) features distinct domains for sequestering differently sized alkanes (as in Nature) as well as internal dimensions suitable for multi-alkane clustering. The ethyl tails of the 29 endohedrally coordinated ligands, L, form a spherical, hydrophobic shell, while their methyl end groups generate a hydrophobic cavity with a diameter of 11 angstrom at the center of the capsule. As such, C-7 to C-3 straight-chain alkanes are tightly intercalated between the ethyl tails, giving assemblies containing 90 to 110 methyl and methylene units, whereas two or three ethane molecules reside in the central cavity of the capsule, where they are free to rotate rapidly, a phenomenon never before observed for the uptake of alkanes from water by molecular cages or containers