The endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo.
ABSTRACT: The LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as 'Ankyrin repeat and LEM domain-containing protein 1' (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.
Project description:Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1?/? mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1?/?-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.
Project description:The GIY-YIG endonuclease family comprises hundreds of diverse proteins and a multitude of functions; none have been visualized bound to DNA. The structure of the GIY-YIG restriction endonuclease R.Eco29kI has been solved both alone and bound to its target site. The protein displays a domain-swapped homodimeric structure with several extended surface loops encircling the DNA. Only three side chains from each protein subunit contact DNA bases, two directly and one via a bridging solvent molecule. Both tyrosine residues within the GIY-YIG motif are positioned in the catalytic center near a putative nucleophilic water; the remainder of the active site resembles the HNH endonuclease family. The structure illustrates how the GIY-YIG scaffold has been adapted for the highly specific recognition of a DNA restriction site, in contrast to nonspecific DNA cleavage by GIY-YIG domains in homing endonucleases or structure-specific cleavage by DNA repair enzymes such as UvrC.
Project description:The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage - possibly by promoting the reorganization of damaged chromatin.
Project description:Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY-YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY-YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.
Project description:The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ???-Me nucleases.
Project description:GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNA-binding domains. I-TevI, the T4 phage GIY-YIG intron endonuclease, functions both in promoting td intron homing, and in acting as a transcriptional autorepressor. Repression is achieved by binding to an operator, which is cleaved at 100-fold reduced efficiency relative to the intronless homing site. The linker includes a zinc finger, which functions in distance determination, to constrain the catalytic domain to cleave the homing site at a fixed position. Here we show that I-BmoI, a related GIY-YIG endonuclease lacking a zinc finger, also possesses some cleavage distance discrimination. Furthermore, hybrid endonucleases constructed by swapping the domains of I-BmoI and I-TevI are active, precise and demonstrate that features other than the zinc finger facilitate distance determination. Most importantly, I-TevI zinc finger mutants cleave the operator more efficiently than the homing site, the converse of wild-type protein. These results are consistent with the zinc finger acting as a measuring device, directing efficient cleavage of the homing site to promote intron mobility, while reducing cleavage at the operator to ensure transcriptional autorepression and phage viability.
Project description:Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.
Project description:The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.
Project description:Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.
Project description:The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.