Conformational Dynamics of Abasic DNA upon Interactions with AP Endonuclease 1 Revealed by Stopped-Flow Fluorescence Analysis

Abstract

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.

The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA

“…Such a difference has occurred due to smaller k −1 value compared to cases of other DNA substrates. According to our previous data [50], AP(2-aPu) substrate has a better structure for catalysis with human AP endonuclease APE1. Thus, His-83 may be responsible for Apn1's specificity to DNA.…”

“…Detection of 2-aPu fluorescence in DNA substrates was used to study the conformational dynamics of damaged DNA during the interaction with H83A Apn1. The fluorescent analog of a heterocyclic base, 2-aPu, was incorporated into the DNA strand in close proximity to the damaged site, as in previous studies [23,25,[50][51][52][53][54].…”

“…With regard to the lesions used, regular AP sites tend to undergo spontaneous β-elimination due to equilibrium formation of an acyclic isomer. To avoid spontaneous substrate degradation, the synthetic analog of abasic site, F, was utilized, as in previous studies [27,29,50,[55][56][57][58][59].…”

“…It was previously shown that modification of bases located at the 5′-side of the AP site had a negative influence on human APE1 activity [50,58]. Since, the AP endonuclease assay using PAGE found no activity of H83A Apn1 on (2-aPu)X duplexes (see Supplementary, Fig.…”

“…Earlier studies on the cleavage of damaged DNA by human BER enzymes, including 8-oxoG DNA glycosylase 1 (hOGG1) and AP endonuclease 1 (APE1), reported that mismatches upstream of the lesion reduced enzymatic activities to a greater extent than downstream ones [50,55,58,60]. The DNA backbone containing an upstream mismatch to AP site was highly bent and distorted in active site of APE1, resulting in a decrease in catalysis rate [50,58]. It should be noted that an upstream mismatch decreases both the affinity of WT APE1 to the damaged DNA and the incision rate of the abasic site F, as compared to a natural authentic AP site [29,50].…”