Search

TREX2, a 3'-5' exonuclease, is a part of the DNA damage tolerance (DDT) pathway that stabilizes replication forks (RFs) by ubiquitinating PCNA along with the ubiquitin E3 ligase RAD18 and other DDT factors. Mismatch repair (MMR) corrects DNA polymerase errors, including base mismatches and slippage. Here we demonstrate that TREX2 deletion reduces mutations in cells upon exposure to genotoxins, including those that cause base lesions and DNA polymerase slippage. Importantly, we show that TREX2 generates most of the spontaneous mutations in MMR-mutant cells derived from mice and people. TREX2-induced mutagenesis is dependent on the nuclease and DNA-binding attributes of TREX2. RAD18 deletion also reduces spontaneous mutations in MMR-mutant cells, albeit to a lesser degree. Inactivation of both MMR and TREX2 additively increases RF stalls, while it decreases DNA breaks, consistent with a synthetic phenotype., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Paired Sp Cas9 nickases ( Sp Cas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired Sp Cas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired Sp Cas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired Sp Cas9n that potentially generate short 3'-overhanging ends at overlapping Sp Cas9n target sites, suggesting sequential nicking of overlapping target sites by Sp Cas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to Sp Cas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to Sp Cas9n. Sp Cas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired Sp Cas9n, allowing practical use of this TREX2-based strategy in genome editing., Competing Interests: A.-Y.X., Y.-L.F., and Y.W. have filed a patent application related to this work., (© 2023 The Author(s).)

Glucose metabolism is known to orchestrate oncogenesis. Whether glucose serves as a signaling molecule directly regulating oncoprotein activity for tumorigenesis remains elusive. Here, we report that glucose is a cofactor binding to methyltransferase NSUN2 at amino acid 1-28 to promote NSUN2 oligomerization and activation. NSUN2 activation maintains global m 5 C RNA methylation, including TREX2, and stabilizes TREX2 to restrict cytosolic dsDNA accumulation and cGAS/STING activation for promoting tumorigenesis and anti-PD-L1 immunotherapy resistance. An NSUN2 mutant defective in glucose binding or disrupting glucose/NSUN2 interaction abolishes NSUN2 activity and TREX2 induction leading to cGAS/STING activation for oncogenic suppression. Strikingly, genetic deletion of the glucose/NSUN2/TREX2 axis suppresses tumorigenesis and overcomes anti-PD-L1 immunotherapy resistance in those cold tumors through cGAS/STING activation to facilitate apoptosis and CD8 + T cell infiltration. Our study identifies NSUN2 as a direct glucose sensor whose activation by glucose drives tumorigenesis and immunotherapy resistance by maintaining TREX2 expression for cGAS/STING inactivation., Competing Interests: Declaration of interests H.-K.L. is a consultant for Stablix, Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)