Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Abstract

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Conformational Dynamics of Biopolymers in the Course of Their Interaction: Multifaceted Approaches to the Analysis by the Stopped-Flow Technique with Fluorescence Detection

“…The kinetic mechanism of conformational changes in APE1 and in abasic DNA molecules in the course of their interaction was studied by recording the intrinsic tryptophan fluorescence of the enzyme [139,140] and the 2-aminopurine fluorescence in the DNA [141]. To elucidate the mechanism of substrate specificity of AP endonucleases, an interaction of enzymes with DNA was analyzed by double electron-electron resonance (DEER, also known as pulsed electron-electron double resonance, PELDOR) spectroscopy [56,142,143] and molecular dynamic simulations [142,144,145], as well as by realtime fluorescent detection of conformational rearrangements of the enzyme and DNA during their interaction [32,33,55,[146][147][148][149][150][151]. The influence of singly (Figure 21a) and doubly (Figure 21b) charged metal ions [152] on APE1's DNA binding and catalysis has been determined.…”

Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1

“…Plasmid pET28c-zAPE1 [10,30] containing the zAPE1 gene carrying an N-terminal His 6 tag was used to construct mutants of the enzyme containing substitution N253G, A254G, or E260A. For expression of the recombinant proteins, 0.5 L of culture of E. coli ArcticExpress (DE3) cells (Invitrogen, Waltham, MA, USA) (in 2xYT broth) carrying the pET28c-zAPE1 construct was grown at 50 µg/mL kanamycin, 50 µg/mL gentamicin, 10 µg/mL tetracycline and 37 • C until absorbance at 600 nm (A 600 ) reached 0.6-0.7.…”

“…Moreover, these DNA adducts are not substrates for any known DNA glycosylase, thereby suggesting that this activity of AP endonucleases is not a "backup" function that is needed in the absence of DNA glycosylases. Additionally, APE1 has 3 -5 -exonuclease activity [7][8][9][10][11][12][13], which is needed in the ATR-Chk1-subpathway of DNA damage response for creating long gaps in DNA [14][15][16][17], and endoribonuclease activity [18][19][20][21][22][23] that was shown to play the role in RNA regulation [24][25][26][27][28].…”

“…Of note, prokaryotic enzyme Xth and human APE1 (hAPE1) share only 27% identity and evolutionarily accumulated changes have led to the absence of NIR activity in Xth [29]. Nonetheless, another study aimed at elucidating the impact of structural differences in homologous APE1s [10,30] has shown that among APE1-like enzymes from humans, D. rerio, Xenopus laevis, and Drosophila melanogaster, only the latter (called Rrp1) has a reduced NIR activity.…”