Abstract
Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.
Highlights
Recognition and removal of non-bulky damaged nitrogenous bases from DNA proceed via the base excision repair (BER) pathway initiated by DNA glycosylases [1,2,3]
We studied real-time conformational changes of the enzymes during their interaction with damaged DNA containing an abasic site (F-site); these changes were monitored by means of changes in Trp fluorescence intensity
Bending and abasic site eversion stages can be determined by a few factors: (i) the formation of network contacts with the DNA backbone upstream and downstream of the lesion, (ii) direct ‘pushing’ or ‘pulling’ interactions with the damaged nucleotide, (iii) possible steric hindrance between the 20 -deoxyribose moiety and active-site amino acid residues in the course of the eversion and (iv) the influence of the complementary-nucleotide nature on the ability of the DNA to bend at the damage site
Summary
Recognition and removal of non-bulky damaged nitrogenous bases from DNA proceed via the base excision repair (BER) pathway initiated by DNA glycosylases [1,2,3]. It can be assumed that non-specific contacts between amino acid residues of the DNAbinding site and DNA backbone serve to sculpt DNA structure for bending the double helix, local DNA melting and damaged-nucleotide eversion. The enzyme-induced distorted state of the DNA helix promotes initial eversion of the damaged nucleotide, and this process may be facilitated by ‘wedging’ interactions with some of active-site amino acid residues. Of note, such non-specific interactions are mostly independent of the nature of the damaged nucleotide and cannot significantly influence the damage recognition process. The comparative analysis of the cleavage of the model DNA substrates by AP endonucleases from Nfo and Xth structural families allowed us to elucidate the mechanism of catalytic-complex formation and to reveal the key steps that affected abasic-site recognition
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