Abstract
Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair enzyme which uses a single active site to process DNA damage via two distinct activities: (1) AP-endonuclease and (2) 3′ to 5′ exonuclease. The AP-endonuclease activity cleaves at AP-sites, while the exonuclease activity excises bulkier 3′ mismatches and DNA damage to generate clean DNA ends suitable for downstream repair. Molecular details of the exonuclease reaction and how one active site can accommodate various toxic DNA repair intermediates remains elusive despite being biologically important. Here, we report multiple high-resolution APE1–DNA structural snapshots revealing how APE1 removes 3′ mismatches and DNA damage by placing the 3′ group within the intra-helical DNA cavity via a non-base flipping mechanism. This process is facilitated by a DNA nick, instability of a mismatched/damaged base, and bending of the DNA. These results illustrate how APE1 cleanses DNA dirty-ends to generate suitable substrates for downstream repair enzymes.
Highlights
Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair enzyme which uses a single active site to process DNA damage via two distinct activities: (1) APendonuclease and (2) 3′ to 5′ exonuclease
The biological significance of APE1 is highlighted by embryonic lethality in mice where its expression is knocked out, expression of inactive variants enhancing cellular sensitivity to DNA damaging therapeutics, and polymorphisms associated with an increased cancer risk[34,35,36]
How a single APE1 active site can have various biological activities on an array of nucleic acid substrates has been of interest to researchers for decades[2,8,11,26,37,38,39]
Summary
APE1 bound to a mismatched DNA exonuclease substrate. We obtained a structure of APE1 engaged with a double stranded a DNA base damage. The APE1 exo substrate structure revealed the mismatched C is in position for removal by incision of the 5′-phosphate backbone opposite an “opposing” T base and a bend in the DNA (Fig. 1c, d). To obtain an APE1 exo product structure, we first grew an exo substrate complex crystal in the presence of CaCl2 and subsequently transferred it to a cryosolution containing MgCl2 to initiate catalysis This approach relies on APE1 to perform backbone cleavage, excising the base, in crystallo. The resulting crystal was flash frozen after soaking for 2.5 h and diffracted to 2.3 Å (Table 1) This structure shows APE1 in complex with its single nucleotide gapped DNA product, revealing the mismatched base has been removed (Fig. 4a). APE1 likely removes a matched base using the same mechanism as a mismatched base, just with a lower efficiency due to the relative lack of flexibility at
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