Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence

Abstract

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3’ exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme’s activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres.

Cleavage of the APE1 N-Terminal Domain in Acute Myeloid Leukemia Cells Is Associated with Proteasomal Activity

“…Among PTMs, APE1 acetylation was highly characterized in terms of structural assignment and consequent biological outcomes. Specifically, acetylated residues (Lys27, Lys31, Lys32 and Lys35) are clustered in the protein N-terminus and are involved in protein-protein interactions [7], APE1 binding to RNAs and DNAs [35], endonuclease activity [5,15,35] and protein binding to nCaRE sequences [36]. Moreover, these residues are ubiquitinated by several ubiquitin ligases (e.g., MDM2, UBR3 and Parkin) [8,37,38], which thus control the steady state level of the protein.…”

“…HeLa cells were transiently transfected with FLAG-tagged APE1 mutants (APE1 WT , APE1 , APE1 K4pleA , APE1 N∆33 ) using the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, and collected 24 h after transfection. Co-immunoprecipitation experiments were performed as already described [15].…”

“…The expression and purification of recombinant proteins from E. coli was performed as previously described [5,15]. When recombinant proteins were used for in vitro assays, the acronym rAPE1 was reported in the text.…”

Metal–Peptide Complexes as Promising Antibiotics to Fight Emerging Drug Resistance: New Perspectives in Tuberculosis

“…Nuclease function is involved in a variety of biological functions, including nucleic acid synthesis, recombination, regulation, processing, and degradation [57][58][59][60], hence the development of low molecular weight metal complexes as "artificial nucleases" is of crucial importance [61]. For most natural metallonucleases, Mg 2+ is the best candidate ion due to its high natural availability and Lewis acidity, but several examples involving Zn 2+ , Ca 2+ , and other metals have been reported [62,63]: in these studies, the metal ion promotes hydrolysis of the nucleic acid phosphate backbone [64].…”

A Dual Face of APE1 in the Maintenance of Genetic Stability in Monocytes: An Overview of the Current Status and Future Perspectives

“…It was found that a loss of APE1 leads to the depletion of TRF2 binding and genomic instability [77]. Burra and co-workers showed that APE1 N-terminal sequences are essential for controlling AP endonuclease enzymatic activity on telomeres' structure [78]. Moreover, APE1 plays a pivotal role in cellular senescence and aging features and it is associated with interaction with sheltering proteins and telomere status [26].…”