Crystal structure of human Fanconi-associated nuclease 1.
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ABSTRACT:
SUBMITTER: Yan PX
PROVIDER: S-EPMC4348246 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI-FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.
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Project description:CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.
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Project description:Fanconi-associated nuclease 1 (FAN1) removes interstrand DNA crosslinks (ICLs) through its DNA flap endonuclease and exonuclease activities. Crystal structures of human and bacterial FAN1 bound to a DNA flap have been solved. The Pseudomonas aeruginosa bacterial FAN1 and human FAN1 (hFAN1) missing a flexible loop are monomeric, while intact hFAN1 is homo-dimeric in structure. Importantly, the monomeric and dimeric forms of FAN1 exhibit very different DNA binding modes. Here, we interrogate the functional differences between monomeric and dimeric forms of FAN1 and provide an explanation for the discrepancy in oligomeric state between the two hFAN1 structures. Specifically, we show that the flexible loop in question is needed for hFAN1 dimerization. While monomeric and dimeric bacterial or human FAN1 proteins cleave a short 5' flap strand with similar efficiency, optimal cleavage of a long 5' flap strand is contingent upon protein dimerization. Our study therefore furnishes biochemical evidence for a role of hFAN1 homodimerization in biological processes that involve 5' DNA Flap cleavage.
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Project description:The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.
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Project description:Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
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Project description:Fanconi Anaemia (FA) is a cancer predisposition disorder characterized by spontaneous chromosome breakage and high cellular sensitivity to genotoxic agents. In response to DNA damage, a multi-subunit assembly of FA proteins, the FA core complex, monoubiquitinates the downstream FANCD2 protein. The FANCE protein plays an essential role in the FA process of DNA repair as the FANCD2-binding component of the FA core complex. Here we report a crystallographic and biological study of human FANCE. The first structure of a FA protein reveals the presence of a repeated helical motif that provides a template for the structural rationalization of other proteins defective in Fanconi Anaemia. The portion of FANCE defined by our crystallographic analysis is sufficient for interaction with FANCD2, yielding structural information into the mode of FANCD2 recruitment to the FA core complex. Disease-associated mutations disrupt the FANCE-FANCD2 interaction, providing structural insight into the molecular mechanisms of FA pathogenesis.
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Project description:The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a "5' end binding pocket" that hosts the 5' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3' flap DNA substrates with 5' nicked ends.
| S-EPMC4193937 | biostudies-literature
Project description:Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2(Ub)) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2(Ub) isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.
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Project description:Mitochondrial RNA polymerase produces long polycistronic precursors that contain the mRNAs, rRNAs and tRNAs needed for mitochondrial translation. Mitochondrial RNase P (mt-RNase P) initiates the maturation of the precursors by cleaving at the 5' ends of the tRNAs. Human mt-RNase P is only active as a tripartite complex (mitochondrial RNase P proteins 1-3; MRPP1-3), whereas plant and trypanosomal RNase Ps (PRORPs)-albeit homologous to MRPP3-are active as single proteins. The reason for this discrepancy has so far remained obscure. Here, we present the crystal structure of human MRPP3, which features a remarkably distorted and hence non-productive active site that we propose will switch to a fully productive state only upon association with MRPP1, MRPP2 and pre-tRNA substrate. We suggest a mechanism in which MRPP1 and MRPP2 both deliver the pre-tRNA substrate and activate MRPP3 through an induced-fit process.
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Project description:Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating innate immune responses against foreign pathogens and other types of dangers through their role in Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and IL-1Rs recruit adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88), to the membrane, which in turn recruit IRAKs via the death domains in these proteins to form the Myddosome complex, leading to IRAK kinase activation. Despite their biological and clinical significance, only the IRAK4 kinase domain structure has been determined among the four IRAK family members. Here, we report the crystal structure of the human IRAK1 kinase domain in complex with a small molecule inhibitor. The structure reveals both similarities and differences between IRAK1 and IRAK4 and is suggestive of approaches to develop IRAK1- or IRAK4-specific inhibitors for potential therapeutic applications. While the IRAK4 kinase domain is capable of homodimerization in the unphosphorylated state, we found that the IRAK1 kinase domain is constitutively monomeric regardless of its phosphorylation state. Additionally, the IRAK1 kinase domain forms heterodimers with the phosphorylated, but not unphosphorylated, IRAK4 kinase domain. Collectively, these data indicate a two-step kinase activation process in which the IRAK4 kinase domain first homodimerizes in the Myddosome, leading to its trans-autophosphorylation and activation. The phosphorylated IRAK4 kinase domain then forms heterodimers with the IRAK1 kinase domain within the Myddosome, leading to its subsequent phosphorylation and activation.
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