TREX1’s Homodimer Structure Has Evolved for Double-Stranded DNA Degradation

The 3’ → 5’ exonuclease TREX1 functions in vivo to degrade DNA and prevent chronic immune activation through the cGAS-STING pathway. TREX1 has been described as a nonprocessive enzyme with little preference for ss-versus dsDNA, raising questions about its principle in vivo substrate. In addition, there has been minimal data rationalizing TREX1’s obligate homodimer structure. We used a combination of biochemical assays, molecular dynamics simulations, and robust kinetic modeling to determine TREX1’s relative affinities for ss-versus dsDNA substrates, to quantify its processivity, and to interrogate the possibility of inter-protomer communication within the TREX1 homodimer. Our findings indicate that TREX1 is a semi-processive exonuclease with a predicted several orders of magnitude greater affinity for dsDNA than ssDNA. Furthermore, we find extensively correlated dynamics between TREX1’s protomers that involve previously described and unrecognized substrate-adjacent regions of the TREX1 enzyme. We infer that TREX1 has evolved as a semi-processive exonuclease that likely degrades dsDNA in vivo, and we propose that its homodimer structure facilitates a mechanism for efficient binding and catabolism of nicked dsDNA. Overall, these studies identify potentially critical regions of the TREX1 enzyme not previously interrogated and contribute to a still-emerging model of TREX1 exonuclease activity.