Mechanism of DNA unwinding by MCM8-9 in complex with HROB.

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.

Short-range translocation by a restriction enzyme motor triggers diffusion along DNA.

Cleavage of bacteriophage DNA by the Type III restriction-modification enzymes requires long-range interaction between DNA sites. This is facilitated by one-dimensional diffusion ('DNA sliding') initiated by ATP hydrolysis catalyzed by a superfamily 2 helicase-like ATPase. Here we combined ultrafast twist measurements based on plasmonic DNA origami nano-rotors with stopped-flow fluorescence and gel-based assays to examine the role(s) of ATP hydrolysis. Our data show that the helicase-like domain has multiple roles. First, this domain stabilizes initial DNA interactions alongside the methyltransferase subunits. Second, it causes environmental changes in the flipped adenine base following hydrolysis of the first ATP. Finally, it remodels nucleoprotein interactions via constrained translocation of a ∼ 5 to 22-bp double stranded DNA loop. Initiation of DNA sliding requires 8-15 bp of DNA downstream of the motor, corresponding to the site of nuclease domain binding. Our data unify previous contradictory communication models for Type III enzymes.

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB.

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB.

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.

Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2.

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by Cells.

The modular construction of Layer-by-Layer biopolymer microcarriers facilitates a highly specific design of drug delivery systems. A supported lipid bilayer (SLB) contributes to biocompatibility and protection of sensitive active agents. The addition of a lipid anchor equipped with PEG (shielding from opsonins) and biotin (attachment of exchangeable outer functional molecules) enhances the microcarrier functionality even more. However, a homogeneously assembled supported lipid bilayer is a prerequisite for a specific binding of functional components. Our investigations show that a tightly packed SLB improves the efficiency of functional components attached to the microcarrier's surface, as illustrated with specific antibodies in cellular application. Only a low quantity of antibodies is needed to obtain improved cellular uptake rates independent from cell type as compared to an antibody-functionalized loosely packed lipid bilayer or directly assembled antibody onto the multilayer. A fast disassembly of the lipid bilayer within endolysosomes exposing the underlying drug delivering multilayer structure demonstrates the suitability of LbL-microcarriers as a multifunctional drug delivery system.

Design of a homogeneous multifunctional supported lipid membrane on layer-by-layer coated microcarriers.

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Development of LbL biopolymer capsules as a delivery system for the multilayer-assembled anti-inflammatory substance α1-antitrypsin.

The treatment of chronic inflammation requires new concepts since recent approaches are mostly accompanied by massive side effects. Layer-by-layer (LbL) microcapsules, functionalized with anti-inflammatory substances such as α1-antitrypsin (AT), may avoid major side- and off-target effects thanks to their local application and the sustained delivery of defined amounts of the active agents into polymorphonuclear leukocytes (PMNs). However, LbL microcapsule application in inflamed tissues requires specific design and preparation. High biocompatibility has to be guaranteed by using biopolymers and ensuring the complete dissolution of the particle core. Moreover, off-target effects - such as macrophage-mediated pro-inflammatory signaling - have to be avoided. In our approach, biopolymer-coated CaCO3 particles were used and the core dissolution process was optimized to obtain highly biocompatible, non-aggregated, long-time stable and clearly calcium-free capsules. A fast verification tool was applied to monitor the remaining Ca2+ content. The incubation with macrophages shows reduced pro-inflammatory signaling compared to microparticles. Regarding their performance as a drug delivery system, AT-functionalized capsules showed a high inhibiting capacity towards neutrophil elastase, a major degradative enzyme in chronic inflammation. Consequently, the optimized design and preparation methods described in this study provide the basis for the development of medically applicable LbL carrier systems for active agents in the treatment of inflammatory processes.

Inhibition of human neutrophil elastase by α1-antitrypsin functionalized colloidal microcarriers.

Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of α(1)-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intra- and extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra- and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.