Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics

Abstract

Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

Multiply Guaranteed Catalytic DNA Circuit for Cancer-Cell-Selective Imaging of miRNA and Robust Evaluation of Drug Resistance

“…Accordingly, the AP-mediated circuitry blockage promotes the stability of the circuitry reactants, thus mitigating the undesirable signal leakage. With the assistance of APE1, the AP site could be specifically recognized and incised to generate strand nicks, 41 Subsequently, the reaction condition was optimized (Figures S4−S6), and the intact AAE system was explored by real-time fluorescence experiment. The enzyme regulation and target stimulation formed a solid basis for efficient fluorescence transduction (Figure 2B).…”

“…Accordingly, the AP-mediated circuitry blockage promotes the stability of the circuitry reactants, thus mitigating the undesirable signal leakage. With the assistance of APE1, the AP site could be specifically recognized and incised to generate strand nicks, thus leading to the detachment of F from BF and facilitating the sensing recovery of S*. Then, these two decaged probes F and S* trigger the downstream signal transducer for motivating the amplified detection of miR-21 in the ST module.…”

Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis

“…The lengths of the dsDNA regions of all P/T DNA substrates are identical (29 bp) and in agreement with the requirements for assembly of a single PCNA onto DNA by RFC ( 21 , 27 , 35 ). The lengths of the template ssDNA regions (33 nt) adjacent to the P/T junctions accommodate 1 RPA at the RPA:DNA ratios utilized ( 12–14 , 36 ).…”

UNG-RPA interaction governs the choice between high-fidelity and mutagenic uracil repair

“…This process uses the complementary strand as a template for repair fidelity. APE1 has low activity on AP sites in the context of ssDNA, and it is known that RPA inhibits APE1 activity in vitro 64,65 . In the context of ssDNA, the RPA WHD domain facilitates UNG access and removal of uracil in ssDNA 66 .…”