Apurinic/apyrimidinic (AP) endonuclease 1 processing of AP sites with 5′ mismatches

Abstract

Despite the DNA duplex being central to biological functions, many intricacies of this molecule, including the dynamic nature of mismatched base pairing, are still unknown. The unique conformations adopted by DNA mismatches can provide insight into the forces at play between nucleotides. Moreover, DNA-binding proteins apply their own individualized steric and electrochemical influences on the nucleotides that they interact with, further altering base-pairing conformations. Here, seven X-ray crystallographic structures of the human nuclease apurinic/apyrimidinic (AP) endonuclease 1 (APE1) in complex with its substrate target flanked by a 5' mismatch are reported. The structures reveal how APE1 influences the conformations of a variety of different mismatched base pairs. Purine-purine mismatches containing a guanine are stabilized by a rotation of the guanine residue about the N-glycosidic bond to utilize the Hoogsteen edge for hydrogen bonding. Interestingly, no rotation of adenine, the other purine, is observed. Mismatches involving both purine and pyrimidine bases adopt wobble conformations to accommodate the mismatch. Pyrimidine-pyrimidine mismatches also wobble; however, the smaller profile of a pyrimidine base results in a gap between the Watson-Crick faces that is reduced by a C1'-C1' compression. These results advance our understanding of mismatched base pairing and the influence of a bound protein.

Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker

“…The resulting APE1:rAP substrate complex reveals a ribo abasic site that is flipped out of the double-helix and into the APE1 active site, as seen previously for deoxy substrates [23] , [38] , [39] . The bridging backbone oxygen, situated 5′ to the abasic site, is near N174, while the non-bridging oxygen and sulfur are coordinated by Y171 and H309.…”

Mechanistic Insight into AP-Endonuclease 1 Cleavage of Abasic Sites at Stalled Replication Forks

“…The resulting APE1:ssDNA product complex reveals a well-formed APE1 active site, as seen previously for dsDNA structures (Figure 5B) 36,[39][40] . A single catalytic Mg 2+…”

AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop

“…We obtained the APE1 Y269A product complex in the presence of MnCl 2. This structure revealed an active site very similar to that of previously reported WT APE1 product structures (Figure 4A ) ( 13 , 14 , 42 ). The abasic site is flipped out of the double helix and into the active site binding pocket where it is in position for cleavage of the phosphate backbone 5′ to the abasic site.…”