Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

Abstract

TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 39!59 activity. This exonuclease is responsible in humans for Aicardi-Goutières syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method, as well as by a new technique developed for monitoring the real-time exonuclease reaction. The X-ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 Å and 2.3 Å , respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the noncatalytic metals competing with distinct affinities for the two metal-binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.Keywords: exonucleases; TREX1; DEDDh family; structure; enzymatic activityThe 39!59 exonucleases play a crucial role in many biological processes by removing nucleotides at the 39 termini from DNA ends, which must then be remodeled in order to prevent the formation of aberrant structures. The 39!59 exonucleases were initially found as proofreading enzymes associated with DNA polymerases. However, in recent years several eukaryotic 39!59 exonucleases, such as p53, the Werner syndrome protein (WRN), and MRE11, 3 These two authors contributed equally to this work.

Characterization of Trex1 Induction by IFN-γ in Murine Macrophages

“…We demonstrate that lithium and sodium inhibit the exonucleolytic activity of Trex1 by inducing subtle rearrangements in active centers. Our analysis also revealed that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of Trex1 (15).…”

“…For quantitative RT-PCR analysis, RNA was treated with DNAse (Roche) to remove contaminating DNA. For cDNA synthesis, 1 mg RNA and Moloney murine leukemia virus RT RNase H Minus, Point Mutant, oligo(dT) 15 primer and PCR nucleotide mix were used, following the manufacturer's instructions (Promega Corporation, Madison, WI). The primers used to amplify murine Trex1 cDNA were 59-CGTCAACGCTTCGATGACA-39 and 59-AGTCATAG-CGGTCACCGTTGT-39.…”

A Conserved Apomixis-Specific Polymorphism Is Correlated with Exclusive Exonuclease Expression in Premeiotic Ovules of Apomictic Boechera Species

“…Enzymes of the DEDD 39→59 exonuclease superfamily (also named the DnaQ superfamily) are characterized by four acidic residues, three Asp (D) and one Glu (E), distributed over three separate sequence segments (Exo I, Exo II, and Exo III; Moser et al, 1997;Zuo and Deutscher, 2001). Residues from this DEDD motif constitute binding sites A and B for two divalent metal ions that are essential for the catalysis of nucleoside monophosphates at DNA or RNA termini in the 39→59 direction (Derbyshire et al, 1995;Brucet et al, 2008). Besides the substrate-binding sites, the presence of either Tyr or His at the active sites defines the DEDDy or DEDDh family, respectively.…”

The Exonuclease Trex1 Restrains Macrophage Proinflammatory Activation

“…TREX1 activity has a propensity toward certain DNA sequences and requires magnesium and manganese in the active site for proper functioning (5). In humans, mutations in TREX1 are associated with the Aicardi-Goutières syndrome (AGS), a neurologic disease characterized by chronic production of IFN in the CNS (6).…”