Human TREX1 Repairs 3′-End DNA Lesions in Vitro

Abstract

Human three-prime repair exonuclease 1 (TREX1) is the major 3′ to 5′ exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3′-phospho-α,β-unsaturated aldehyde and 3′-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

PARP1 Incises Abasic Sites and Covalently Cross-links to 3′-DNA Termini via Cysteine Addition Not Reductive Amination

“…3′-PUA was prepared as a control by heating the AP site as previously described. 17 We found that nearly all nonreduced DPCs (Figure 2B proposed 3′-PUA-PARP1 DPC structure (Figure 1). This also indicates that the initially formed Schiff base 3′-PUA-PARP1 DPC intermediate is hydrolyzed before or after the Michael addition.…”

“…Specifically, the isolated PARP1-DPC without or with prior reduction by NaBH 4 was reacted with ARP, followed by proteinase K treatment and urea-PAGE analysis. 3′-PUA was prepared as a control by heating the AP site as previously described . We found that nearly all nonreduced DPCs (Figure B, Lanes 5–6), but not the reduced DPCs (Figure B, Lanes 1–2), were converted to slower migrating products (i.e., proteolyzed 3′-PUA-PARP1-ARP).…”

Synthesis and Excision Repair of Site-Specific 3′-End DNA-Histone Cross-Links Derived from Abasic Sites

“…We did a control experiment and demonstrated that this has little effect on the repair. Specifically, an oligonucleotide (Table S1, O4) containing 6-carboxyfluorescein (6-FAM) at the 5′-terminus and 3′-PUA was prepared as previously described 18 and then reacted with O-2propynylhydroxylamine 19 (AA3, Figure 2A). The resulting 3′-PUA-alkyne was purified by urea-PAGE, and the correct molecular weight was confirmed by MALDI-TOF mass spectrometry (Figure S1).…”