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      The hSNM1 protein is a DNA 5′-exonuclease

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          Abstract

          The human SNM1 protein is a member of a highly conserved group of proteins catalyzing the hydrolysis of nucleic acid substrates. Although overproduction is unstable in mammalian cells, we have overproduced a recombinant hSNM1 protein in an insect cell system. The protein is a single-strand 5′-exonuclease, like its yeast homolog. The enzyme utilizes either DNA or RNA substrates, requires a 5′-phosphate moiety, shows very little activity on double-strand substrates, and functions at a size consistent with a monomer. The exonuclease activity requires the conserved β-lactamase domain; site-directed mutagenesis of a conserved aspartate inactivates the exonuclease.

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          Most cited references28

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          Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination.

          Mutations in the Artemis protein in humans result in hypersensitivity to DNA double-strand break-inducing agents and absence of B and T lymphocytes (radiosensitive severe combined immune deficiency [RS-SCID]). Here, we report that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in the absence of DNA. The purified Artemis protein alone possesses single-strand-specific 5' to 3' exonuclease activity. Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on 5' and 3' overhangs, as well as hairpins. Finally, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex. Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical for the hairpin-opening step of V(D)J recombination and for the 5' and 3' overhang processing in nonhomologous DNA end joining.
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            Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency.

            The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.
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              Metallo-beta-lactamase fold within nucleic acids processing enzymes: the beta-CASP family.

              A separate family of enzymes within the metallo-beta-lactamase fold comprises several important proteins acting on nucleic acid substrates, involved in DNA repair (Artemis, SNM1 and PSO2) and RNA processing [cleavage and polyadenylation specificity factor (CPSF) subunit]. Proteins of this family, named beta-CASP after the names of its representative members, possess specific features relative to those of other metallo-beta-lactamases, that are concentrated in the C-terminal part of the domain. In this study, using sensitive methods of sequence analysis, we identified highly conserved amino acids specific to the beta-CASP family, some of which were unidentified to date, that are predicted to play critical roles in the enzymatic function. The identification and characterisation of all the extant, detectable beta-CASP members within sequence databases and genome data also allowed us to unravel particular sequence features which are likely to be involved in substrate specificity, as well as to describe new but as yet uncharacterised members which may play critical roles in DNA and RNA metabolism.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                September 2007
                5 September 2007
                4 September 2007
                : 35
                : 18
                : 6115-6123
                Affiliations
                Oregon Health & Science University, Department of Molecular and Medical Genetics, 3181 SW Sam Jackson Park Road, Mail Code L103, Portland, OR 97239-3098
                Author notes
                *To whom correspondence should be addressed. +503 494 6881+503 494 6886 mosesr@ 123456ohsu.edu
                Article
                10.1093/nar/gkm530
                2094091
                17804464
                4e5a7d42-1d45-4f7e-ae16-555992e83291
                © 2007 The Author(s)

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 30 April 2007
                : 20 June 2007
                : 26 June 2007
                Categories
                Nucleic Acid Enzymes

                Genetics
                Genetics

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