Structure of the Dimeric Exonuclease TREX1 in Complex with DNA Displays a Proline-rich Binding Site for WW Domains

Abstract

TREX1 is the most abundant mammalian 3 3 5 DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Coimmunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

The TREX1 Double-stranded DNA Degradation Activity Is Defective in Dominant Mutations Associated with Autoimmune Disease

“…We showed previously that TREX1 removes nucleotides from ssDNA and dsDNA oligonucleotide constructs (1,3,5) and from a nicked dsDNA plasmid generated by incubation with the NM23-H1 endonuclease (6). The TREX1 structure indicates that four nucleotides of ssDNA, not dsDNA, bind into the active sites of each protomer of the dimer (9,22) consistent with the greater TREX1 activity detected on partial duplex DNAs containing 3Ј terminal unpaired nucleotides (1,3). These combined data illustrate TREX1 exonuclease action using a variety of DNA structures that contain accessible 3Ј termini.…”

TREX1 DNA exonuclease deficiency, accumulation of single stranded DNA and complex human genetic disorders

“…PPII refers to the polyproline II motif implicated in mediating protein-protein interactions with proline-binding domain containing proteins. In fact, PPII has been shown to be required for the interaction of Trex1 with the transcription factor CA150 [16]. The functional significance of this interaction remains to be clarified.…”

“…Trex1 functions as a homodimer and its crystal structure has recently been solved [16,17]. For a detailed discussion of Trex1 structure-function analysis readers are referred to the studies by Brucet and colleagues and de Silva and colleagues [16,17]. Exo 1-3 represent the exonuclease domains which co-ordinate Mg +2 which is essential for catalysis.…”

“…exonuclease activity but impact on Trex1 intra-cellular localisation [52]. Work on TREX1 is currently uncovering novel interacting proteins and should provide more information on the multifunctional nature of this enzyme [16]. Finally, it will be important to uncover the extent of functional overlap, if any, between TREX1 and Trex2.…”

“…The precise biological function of TREX2 is currently unclear although a possible role in mediating genomic stability has been suggested [15]. The crystal structure of Trex1 has recently been described and readers are referred to these studies for in-depth description of TREX1 structure-function analysis [16,17].…”