ABSTRACT
The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Endonucleases/chemistry , Endonucleases/metabolism , Binding Sites , Cryoelectron Microscopy , DNA-Binding Proteins/genetics , Endonucleases/genetics , Fanconi Anemia/enzymology , Fanconi Anemia/genetics , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Domains , Protein Multimerization , Structure-Activity Relationship , Xeroderma Pigmentosum/enzymology , Xeroderma Pigmentosum/geneticsABSTRACT
Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling.
Subject(s)
DNA Helicases/chemistry , DNA-Binding Proteins/chemistry , Adenosine Triphosphate/chemistry , Catalytic Domain , Cell Survival/drug effects , Coordination Complexes/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , DNA Damage , DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fanconi Anemia Complementation Group Proteins , Gene Knockdown Techniques , HEK293 Cells , Humans , Hydrolysis , Mitomycin/pharmacology , Models, Molecular , Mutagens/pharmacology , Nucleic Acid Conformation , Oligonucleotides/chemistry , Protein Binding , Protein Structure, Secondary , RNA, Small Interfering/geneticsABSTRACT
The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.
Subject(s)
DNA-Binding Proteins/chemistry , Endodeoxyribonucleases/metabolism , Endonucleases/chemistry , Amino Acid Sequence , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Humans , Molecular Sequence Data , Mutation , Protein Structure, TertiaryABSTRACT
The structure-specific endonuclease activity of the human XPF-ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF-ERCC1 cleaves DNA structures such as stem-loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem-loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF-ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3'-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix-hairpin-helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF-HLD shows a preference for binding stem-loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF-ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.
