ABSTRACT
p97 is a highly abundant, homohexameric AAA+ ATPase that performs a variety of essential cellular functions. Characterized as a ubiquitin-selective chaperone, p97 recognizes proteins conjugated to K48-linked polyubiquitin chains and promotes their removal from chromatin and other molecular complexes. Changes in p97 expression or activity are associated with the development of cancer and several related neurodegenerative disorders. Although pathogenic p97 mutations cluster in and around p97's ATPase domains, mutant proteins display normal or elevated ATPase activity. Here, we show that one of the most common p97 mutations (R155C) retains ATPase activity, but is functionally defective. p97-R155C can be recruited to ubiquitinated substrates on chromatin, but is unable to promote substrate removal. As a result, p97-R155C acts as a dominant negative, blocking protein extraction by a similar mechanism to that observed when p97's ATPase activity is inhibited or inactivated. However, unlike ATPase-deficient proteins, p97-R155C consumes excess ATP, which can hinder high-energy processes. Together, our results shed new insight into how pathogenic mutations in p97 alter its cellular function, with implications for understanding the etiology and treatment of p97-associated diseases.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Adenosine Triphosphatases/chemistry , Amino Acid Substitution , Animals , Cell Line, Tumor , Chromatin/metabolism , DNA/metabolism , Female , Humans , In Vitro Techniques , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Neoplasms/enzymology , Neoplasms/genetics , Nuclear Proteins/chemistry , Oocytes/metabolism , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Ubiquitin/metabolism , Xenopus laevisABSTRACT
DNA crosslinking agents make up a broad class of chemotherapy agents that target rapidly dividing cancer cells by disrupting DNA synthesis. These drugs differ widely in both chemical structure and biological effect. In cells, crosslinking agents can form multiple types of DNA lesions with varying efficiencies. Inter-strand crosslinks (ICLs) are considered to be the most cytotoxic lesion, creating a covalent roadblock to replication and transcription. Despite over 50 years in the clinic, the use of crosslinking agents that specialize in the formation of ICLs remains limited, largely due to high toxicity in patients. Current ICL-based therapeutics have focused on late-stage and drug-resistant tumors, or localized treatments that limit exposure. In this article, we review the development of clinical crosslinking agents, our understanding of how cells respond to different lesions, and the potential to improve ICL-based chemotherapeutics in the future.
Subject(s)
Antineoplastic Agents/therapeutic use , Cross-Linking Reagents/therapeutic use , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Cross-Linking Reagents/pharmacology , DNA/drug effects , Furocoumarins/pharmacology , Furocoumarins/therapeutic use , Humans , Mechlorethamine/analogs & derivatives , Mechlorethamine/therapeutic use , Mitomycins/pharmacology , Mitomycins/therapeutic useABSTRACT
Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying cross-link. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , DNA Helicases/metabolism , DNA Repair , Xenopus laevis/genetics , Animals , Chromatin/enzymology , DNA Replication , Ubiquitination , Valosin Containing Protein , Xenopus Proteins/metabolism , Xenopus laevis/metabolismABSTRACT
The relationship between chemical structure and gelation ability was examined for a series of nine Hg-containing compounds. Both solid-state properties (dissolution enthalpies/entropies and packing structure) and gel properties (strength, morphology, cation selectivity, and anion tolerance) were examined. Overall, the results reveal a complex relationship between chemical structure and properties. The remediation potential of these Hg-triggered gelations was also investigated, revealing that >98% of the Hg(2+) in water can be removed through gel formation.
