Structural basis for overhang excision and terminal unwinding of DNA duplexes by TREX1

Abstract

Three prime repair exonuclease 1 (TREX1) is an essential exonuclease in mammalian cells, and numerous in vivo and in vitro data evidenced its participation in immunity regulation and in genotoxicity remediation. In these very complicated cellular functions, the molecular mechanisms by which duplex DNA substrates are processed are mostly elusive because of the lack of structure information. Here, we report multiple crystal structures of TREX1 complexed with various substrates to provide the structure basis for overhang excision and terminal unwinding of DNA duplexes. The substrates were designed to mimic the intermediate structural DNAs involved in various repair pathways. The results showed that the Leu24-Pro25-Ser26 cluster of TREX1 served to cap the nonscissile 5′-end of the DNA for precise removal of the short 3′-overhang in L- and Y-structural DNA or to wedge into the double-stranded region for further digestion along the duplex. Biochemical assays were also conducted to demonstrate that TREX1 can indeed degrade double-stranded DNA (dsDNA) to a full extent. Overall, this study provided unprecedented knowledge at the molecular level on the enzymatic substrate processing involved in prevention of immune activation and in responses to genotoxic stresses. For example, Arg128, whose mutation in TREX1 was linked to a disease state, were shown to exhibit consistent interaction patterns with the nonscissile strand in all of the structures we solved. Such structure basis is expected to play an indispensable role in elucidating the functional activities of TREX1 at the cellular level and in vivo.

Structural insights into the duplex DNA processing of TREX2

“…Interestingly, mTREX2 was capable of digesting the double-stranded substrate at a higher concentration (0.4 μM) (Figure 1B). Our results showed that TREX2 exhibits the single-stranded DNA-specific activity at a lower concentration and possesses both single- and double-stranded DNA processing activity at a higher concentration that is similar to those of Exo X (21) and TREX1 (16,21), the E. coli and human homologs of TREX2 (Supplementary Figure S1). The unique catalytic properties of TREX2, Exo X and TREX1 are different from those of other DEDDh exonucleases, such as E. coli RNase T (16,33) which cannot deconstruct the double-stranded DNA structure at the higher concentration of 3 μM (Supplementary Figure S1).…”

“…The structure of TREX2 for binding to duplex DNA with long double-stranded region; thus, TREX2 is suitable for participating in the process of duplex DNA with a longer double-stranded region, such as chromosomal rearrangement and maintenance. The interacting region between TREX1 and duplex DNA focuses on the terminal of duplex DNA; thus, TREX1 fits for dsDNA terminal deconstruction in immune controlling and trimming of structural DNAs in various DNA repair pathways (16). In summary, our results highlight the characteristic of TREX2 on the processing of duplex DNA with long double-stranded region.…”

“…Based on the substrate preference measured by in vitro activity assays, we divided the DEDDh exonucleases into three groups, including ssDNA/ssRNA-specific (43–47), dsDNA/dsRNA-specific (48–50) and duplex DNA/RNA with 3′-overhang-specific DEDDh exonucleases (16,18,22,39,51–53) (Figures 6 and 7). The 3′-overhang-specific DEDDh exonucleases usually contain a narrow active site, and use two hydrophobic resides to stack with the last base at 3′-end (33,54).…”

“…Most members exhibit preferences for the substrate sequence, length and structure. Among the DEDDh exonucleases, TREX1 (16), TREX2 (2,17) and 3′hExo in mammalian cells (18), Snp in Drosophila (19), TTHB178 in Thermus thermophilus (20), as well as Exo X (21,22) and RNase T (21) in Escherichia coli display a similar substrate preference with the duplex DNA/RNA with 3′-overhang; however, their cellular functions are different. Thus, it is challenging to classify the cellular functions of the DEDDh exonucleases based on the substrate preference and in vitro activity assays.…”

A Novel Mutation in Aicardi–Goutières' Syndrome: A Case Report

“…16 Researchers have noted that mutations in TREX1 gene are associated with several diseases, including AGS, FCL, systemic lupus erythematosus (SLE), retinal vasculopathy with cerebral leukodystrophy, Cree encephalitis, and cryofibrinogenemia. 14,17,18 In 2009, Crow and Rehwinkel evaluated the pathogenesis of AGS for the phenotypic overlap with the manifestations of congenital infection and SLE where type I IFN-mediated immunity is provided by viral and self-nucleic acids, Fig. 3 The frameshift mutation in the exon 2 (c.404_407delCAGC) or (p.Ala81AlafsX6) on TREX1 gene makes an early termination in amino acid production, which would be expected to affect the protein's function.…”