A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family.

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition.

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

Systemic lupus erythematosus overlapping dermatomyositis owing to a heterozygous TREX1 Asp130Asn missense mutation.

The 3' repair exonuclease 1 (TREX1) gene encodes a nuclear protein with 3' exonuclease activity, and the mutations have been associated with autoimmune diseases. Herein, we performed genetic analysis for the TREX1 gene in 55 patients with systemic lupus erythematosus (SLE). We identified one SLE patient with overlapping dermatomyositis having a heterozygous p.Asp130Asn mutation in the TREX1 gene. The patient had a high level of serum interferon (IFN)-α compared with that in healthy controls and other patients with SLE. In addition, the patient expressed elevated IFN signature genes compared with healthy controls. Our molecular dynamics simulation of the TREX1 protein in a complex with double-stranded DNA revealed that the D130N mutant causes significant changes in the active site's interaction network. One of our cases exhibited a heterozygous TREX1 p.Asp130Asn mutation that contributed to the type I IFN pathway, which may lead to the development of a severe SLE phenotype.

High-fidelity DNA replication in Mycobacterium tuberculosis relies on a trinuclear zinc center.

High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.

RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity.

Helicases are molecular motors that move along and unwind double-stranded nucleic acids. RecBCD enzyme is a complex helicase and nuclease, essential for the major pathway of homologous recombination and DNA repair in Escherichia coli. It has sets of helicase motifs in both RecB and RecD, two of its three subunits. This rapid, highly processive enzyme unwinds DNA in an unusual manner: the 5'-ended strand forms a long single-stranded tail, whereas the 3'-ended strand forms an ever-growing single-stranded loop and short single-stranded tail. Here we show by electron microscopy of individual molecules that RecD is a fast helicase acting on the 5'-ended strand and RecB is a slow helicase acting on the 3'-ended strand on which the single-stranded loop accumulates. Mutational inactivation of the helicase domain in RecB or in RecD, or removal of the RecD subunit, altered the rates of unwinding or the types of structure produced, or both. This dual-helicase mechanism explains how the looped recombination intermediates are generated and may serve as a general model for highly processive travelling machines with two active motors, such as other helicases and kinesins.

The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.

The Mre11 complex (Mre11 Rad50 Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid association. These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 A crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn(2+) ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 A. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.

Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1.

All organisms express dedicated repair enzymes for counteracting the cytotoxic and mutagenic potential of apurinic/apyrimidinic (AP) lesions, which would otherwise pose a serious threat to genome integrity. We present the predicted three-dimensional structure of the major human AP site-specific DNA repair endonuclease, HAP1, and show that an aspartate/histidine pair, in conjunction with a metal ion-coordinating glutamate residue, are critical for catalyzing the multiple repair activities of HAP1. We suggest that this catalytic mechanism is conserved in certain reverse transcriptases, but is distinct from the two metal ion-mediated mechanism defined for other hydrolytic nucleases.

A multifunctional exonuclease from vegetative Schizosaccharomyces pombe cells exhibiting in vitro strand exchange activity.

A 140-kDa polypeptide (p140) has been purified over 2000-fold from vegetative Schizosaccharomyces pombe cells using an assay of homologous pairing and strand exchange between linear double-stranded DNA (dsDNA) and circular single-stranded DNA (ssDNA) in vitro. Electron microscopic analysis of the reaction products showed displacement of one strand of the linear duplex DNA by the circular ssDNA molecule. In addition, the protein contained 5' to 3' exonuclease activity on ssDNA and dsDNA (with a 50-fold preference on the single-stranded substrate) as well as on single-stranded RNA. Furthermore, p140 was capable of renaturing complementary ssDNA as shown by S1 nuclease assays. p140 behaved like a monomer in solution under reaction conditions. Direct comparison of the biochemical properties, sequence analysis, and cross-reactivity to a monoclonal antibody suggests that p140 is probably identical with ExoII, purified from S. pombe meiotic cells as a ssDNA exonuclease (Szankasi, P., and Smith, G. R. (1992) Biochemistry 31, 6769-6773). Given the diverse activities of p140, the protein might be involved in DNA and/or RNA metabolism in vivo.