ABSTRACT
The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode.
Subject(s)
DNA Helicases/metabolism , Escherichia coli Proteins/metabolism , Rec A Recombinases/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Binding Sites , DNA/metabolism , DNA, Single-Stranded/metabolism , Rec A Recombinases/antagonists & inhibitors , Rec A Recombinases/chemistry , Rec A Recombinases/ultrastructure , Sequence DeletionABSTRACT
Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.
Subject(s)
Benzimidazoles/chemistry , Benzimidazoles/pharmacology , HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/metabolism , Nipecotic Acids/chemistry , Nipecotic Acids/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Allosteric Regulation/drug effects , Animals , Benzimidazoles/metabolism , Benzimidazoles/pharmacokinetics , Biological Availability , Caspases/metabolism , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , HEK293 Cells , HSP70 Heat-Shock Proteins/chemistry , Humans , Mice , Models, Molecular , Nipecotic Acids/metabolism , Nipecotic Acids/pharmacokinetics , Permeability , Piperidines/metabolism , Piperidines/pharmacokinetics , Protein Aggregates/drug effects , Protein Structure, Tertiary , Xenograft Model Antitumor AssaysABSTRACT
The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding protein. The ATPase function of DrRecA nucleoprotein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans.
