Structural Biochemistry and Interaction Architecture of the DNA Double-Strand Break Repair Mre11 Nuclease and Rad50-ATPase

Hopfner, Karl‐Peter; Karcher, Annette; Craig, Lisa; Woo, Tammy T.; Carney, James; Tainer, John A.

doi:10.1016/s0092-8674(01)00335-x

Cited by 466 publications

(597 citation statements)

References 56 publications

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“…This complex contains a bifunctional DNA binding domain in which the active site of the Mre11 nuclease is capable of binding ssDNA, whereas an adjacent site accommodates double-stranded DNA. Occupancy of the sites is mutually exclusive, suggesting that a given interaction with DNA facilitates enzymatic (in the ssDNA binding domain) or structural (in the dsDNA binding domain) functions (Hopfner et al 2001). The data presented here argue that the essential functions of the complex cannot be clearly divided according to its enzymatic or structural roles.…”

Section: The Mre11 Complex: Diverse Mechanisms In Maintaining Genomicmentioning

confidence: 76%

Cancer predisposition and hematopoietic failure in Rad50^S/S mice

Bender¹,

Sikes²,

Sullivan³

et al. 2002

Genes Dev.

187

182

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Mre11, Rad50, and Nbs1 function in a protein complex that is central to the metabolism of chromosome breaks. Null mutants of each are inviable. We demonstrate here that hypomorphic Rad50 mutant mice (Rad50 S/S mice) exhibited growth defects and cancer predisposition. Rad50 S/S mice died with complete bone marrow depletion as a result of progressive hematopoietic stem cell failure. Similar attrition occurred in spermatogenic cells. In both contexts, attrition was substantially mitigated by p53 deficiency, whereas the tumor latency of p53 −/− and p53 +/− animals was reduced by Rad50 S/S . Indices of genotoxic stress and chromosomal rearrangements were evident in Rad50 S/S cultured cells, as well as in Rad50 S/S and p53 −/− Rad50 S/S lymphomas, suggesting that the Rad50 S/S phenotype was attributable to chromosomal instability. These outcomes were not associated with overt defects in the Mre11 complex's previously established double strand break repair and cell cycle checkpoint regulation functions. The data indicate that even subtle perturbation of Mre11 complex functions results in severe genotoxic stress, and that the complex is critically important for homeostasis of proliferative tissues.

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Section: The Mre11 Complex: Diverse Mechanisms In Maintaining Genomicmentioning

confidence: 76%

Cancer predisposition and hematopoietic failure in Rad50^S/S mice

Bender¹,

Sikes²,

Sullivan³

et al. 2002

Genes Dev.

187

182

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“…1II7 is a P. furiosus mre11 nuclease responsible for the DNA double-strand break repair (23). Although the overall sequence homology between MJ0936 and 1II7 is low (19% identity), the active site residues are well conserved in both proteins as shown in Fig.…”

Section: Resultsmentioning

confidence: 98%

Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

Chen

Yakunin

Kuznetsova

et al. 2004

Journal of Biological Chemistry

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Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-Å resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two ␤-sheets flanked by ␣-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni 2؉ or Mn 2؉ produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5 -monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni 2؉ and Mn 2؉ being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.Several hundred genomes have been sequenced so far, but no function can be inferred from the gene sequence of about half of the genes. Structural information may provide new clues as to the molecular (biochemical and biophysical) functions of the proteins coded by these genes of unknown functions.An open reading frame from Methanococcus jannaschii, MJ0936 (GI 1499771), is annotated as a conserved hypothetical protein in the PEDANT sequence data base (1). A PSI-PHI BLAST search found 56 bacterial or archaeal homologs with E value less than 0.003. Some of the homologs are annotated, and they can be grouped into three categories: (i) (conserved) hypothetical protein; (ii) VPS29-like (human vacuolar protein-sorting protein) phosphoesterase-related protein; (iii) predicted/ putative/probable phosphoesterase. Similarly, a Pfam (2) search suggests that this gene may belong to a protein family of calcineurin-like phosphoesterase. This Pfam protein family contains a large range of phosphoesterases, including protein phosphoserine phosphatases, nucleotidases, sphingomyelin phosphodiesterases, 2Ј,3Ј-cyclic AMP phosphodiesterases, and nucleases such as bacterial SbcD or yeast MRE11, as well as the VPS29-like phosphoesterase-related proteins mentioned above. Presumed metal-chelating residues are found to be conserved for the proteins of this family.To determine the function of MJ0936 and its homologues, we have solved the crystal structure of this protein and performed a series of biochemical screenings for catalytic activity. Through a combination of structural and enzymatic analyses, we ...

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“…[10][11][12] Structural comparison to other enzymes indicates that GpdQ belongs to the group of R/ sandwich metallophosphoesterases, which also includes purple acid phosphatases (PAPs), the 3′-5′ cyclic nucleotide diesterase Rv0805 from Mycobacterium tuberculosis, and Mre11 nuclease. [13][14][15][16][17][18][19][20][21][22][23] The active site ( ion in the R site is coordinated by four amino acid residues, a terminal water ligand, and a hydr(oxide) molecule that bridges the two metal ions. In the site, the metal ion may also be coordinated by four amino acid residues but, based on spectroscopic and kinetic data, is predicted to be less tightly bound ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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Abstract:The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K d values for the metal ions in the R and sites to be 29 and 344 µM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the site increases substantially (K d ) 56 µM), while that of the R site is not greatly affected (K d ) 17 µM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k obs1 ), the assembly of a catalytically active binuclear center (k obs2 ), and subsequent slower structural rearrangements to optimize catalysis (k obs3 ). These three phases depend on the concentration of substrate ([S]), with k obs1 and k obs2 reaching maximum values at high [S] (354 and 38 s -1 , respectively), whereas k obs3 is reduced as [S] is increased. The k cat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s -1 ) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k obs3 is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate-and metal-ion-induced fit to optimize catalysis.

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Structural Biochemistry and Interaction Architecture of the DNA Double-Strand Break Repair Mre11 Nuclease and Rad50-ATPase

Cited by 466 publications

References 56 publications

Cancer predisposition and hematopoietic failure in Rad50^S/S mice

Cancer predisposition and hematopoietic failure in Rad50^S/S mice

Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

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Structural Biochemistry and Interaction Architecture of the DNA Double-Strand Break Repair Mre11 Nuclease and Rad50-ATPase

Cited by 466 publications

References 56 publications

Cancer predisposition and hematopoietic failure in Rad50S/S mice

Cancer predisposition and hematopoietic failure in Rad50S/S mice

Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

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Cancer predisposition and hematopoietic failure in Rad50^S/S mice

Cancer predisposition and hematopoietic failure in Rad50^S/S mice