Molecular insight into the specific enzymatic properties of TREX1 revealing the diverse functions in processing RNA and DNA/RNA hybrids.

In various autoimmune diseases, dysfunctional TREX1 (Three prime Repair Exonuclease 1) leads to accumulation of endogenous single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and DNA/RNA hybrids in the cytoplasm and triggers immune activation through the cGAS-STING pathway. Although inhibition of TREX1 could be a useful strategy for cancer immunotherapy, profiling cellular functions in terms of its potential substrates is a key step. Particularly important is the functionality of processing DNA/RNA hybrids and RNA substrates. The exonuclease activity measurements conducted here establish that TREX1 can digest both ssRNA and DNA/RNA hybrids but not dsRNA. The newly solved structures of TREX1-RNA product and TREX1-nucleotide complexes show that 2'-OH does not impose steric hindrance or specific interactions for the recognition of RNA. Through all-atom molecular dynamics simulations, we illustrate that the 2'-OH-mediated intra-chain hydrogen bonding in RNA would affect the binding with TREX1 and thereby reduce the exonuclease activity. This notion of higher conformational rigidity in RNA leading TREX1 to exhibit weaker catalytic cleavage is further validated by the binding affinity measurements with various synthetic DNA-RNA junctions. The results of this work thus provide new insights into the mechanism by which TREX1 processes RNA and DNA/RNA hybrids and contribute to the molecular-level understanding of the complex cellular functions of TREX1 as an exonuclease.

Understanding APE1 cellular functions by the structural preference of exonuclease activities.

Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) has versatile enzymatic functions, including redox, endonuclease, and exonuclease activities. APE1 is thus broadly associated with pathways in DNA repair, cancer cell growth, and drug resistance. Unlike its AP site-specific endonuclease activity in Base excision repair (BER), the 3'-5' exonucleolytic cleavage of APE1 using the same active site exhibits complex substrate selection patterns, which are key to the biological functions. This work aims to integrate molecular structural information and biocatalytic properties to deduce the substrate recognition mechanism of APE1 as an exonuclease and make connection to its diverse functionalities in the cell. In particular, an induced space-filling model emerges in which a bridge-like structure is formed by Arg177 and Met270 (RM bridge) upon substrate binding, causing the active site to adopt a long and narrow product pocket for hosting the leaving group of an AP site or the 3'-end nucleotide. Rather than distinguishing bases as other exonucleases, the hydrophobicity and steric hindrance due to the APE1 product pocket provides selectivity for substrate structures, such as matched or mismatched blunt-ended dsDNA, recessed dsDNA, gapped dsDNA, and nicked dsDNA with 3'-end overhang shorter than 2 nucleotides. These dsDNAs are similar to the native substrates in BER proofreading, BER for trinucleotide repeats (TNR), Nucleotide incision repair (NIR), DNA single-strand breaks (SSB), SSB with damaged bases, and apoptosis. Integration of in vivo studies, in vitro biochemical assays, and structural analysis is thus essential for linking the APE1 exonuclease activity to the specific roles in cellular functions.

APE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling.

The exonuclease activity of Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for processing matched/mismatched terminus in various DNA repair pathways and for removing nucleoside analogs associated with drug resistance. To fill in the gap of structural basis for exonucleolytic cleavage, we determine the APE1-dsDNA complex structures displaying end-binding. As an exonuclease, APE1 does not show base preference but can distinguish dsDNAs with different structural features. Integration with assaying enzyme activity and binding affinity for a variety of substrates reveals for the first time that both endonucleolytic and exonucleolytic cleavage can be understood by an induced space-filling model. Binding dsDNA induces RM (Arg176 and Met269) bridge that defines a long and narrow product pocket for exquisite machinery of substrate selection. Our study paves the way to comprehend end-processing of dsDNA in the cell and the drug resistance relating to APE1.

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

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.