ABSTRACT
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.
Subject(s)
DNA Repair , DNA-Binding Proteins , Minichromosome Maintenance Proteins , Humans , Adenosine Triphosphate/metabolism , DNA/metabolism , DNA/chemistry , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Minichromosome Maintenance Proteins/metabolism , Minichromosome Maintenance Proteins/genetics , Protein Binding , Protein Multimerization , DNA Repair/geneticsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Due to their excellent biocompatibility and biodegradability, natural hydrogels are highly demanded biomaterials for biomedical applications such as wound dressing, tissue engineering, drug delivery or three dimensional cell culture. Highly energetic electron irradiation up to 10â¯MeV is a powerful and fast tool to sterilize and tailor the material's properties. In this study, electron radiation treatment of agarose hydrogels was investigated to evaluate radiation effects on physical, structural and chemical properties. The viscoelastic behavior, surface hydrophilicity and swelling behavior in a range of typical sterilization doses of 0â¯kGy to 30â¯kGy was analyzed. The mechanical properties were determined by rheology measurements and decreased by more than 20% compared to the initial moduli. The number average molecular weight between crosslinks was estimated based on rubber elasticity theory to judge on the radiation degradation. In this dose range, the number average molecular weight between crosslinks increased by more than 6%. Chemical structure was investigated by FTIR spectroscopy to evaluate the radiation resistance of agarose hydrogels. With increasing electron dose, an increasing amount of carbonyl containing species was observed. In addition, irradiation was accompanied by formation of gas cavities in the hydrogels. The gas products were specified for CO2, CO and H2O. Based on the radiolytic products, a radiolysis mechanism was proposed. Electron beam treatment under high pressure conditions was found to reduce gas cavity formation in the hydrogels.
