Modeling of Escherichia coli Endonuclease V structure in complex with DNA

Karolina A. Majorek 0 Janusz M. Bujnicki 0 0 J. M. Bujnicki Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology , Trojdena 4, 02-109 Warsaw, Poland 1 ) Institute for Molecular Biology and Biotechnology, Adam Mickiewicz University , Umultowska 89, PL-61-614 Poznan, Poland Endonuclease V (EndoV) is a metal-dependent DNA repair enzyme involved in removal of deaminated bases (e.g., deoxyuridine, deoxyinosine, and deoxyxanthosine), with pairing specificities different from the original bases. Homologs of EndoV are present in all major phyla from bacteria to humans and their function is quite well analyzed. EndoV has been combined with DNA ligase to develop an enzymatic method for mutation scanning and has been engineered to obtain variants with different substrate specificities that serve as improved tools in mutation recognition and cancer mutation scanning. However, little is known about the structure and mechanism of substrate DNA binding by EndoV. Here, we present the results of a bioinformatic analysis and a structural model of EndoV from Escherichia coli in complex with DNA. The structure was obtained by a combination of fold-recognition, comparative modeling, de novo modeling and docking methods. The modeled structure provides a convenient tool to study protein sequence-structure-function relationships in EndoV and to engineer its further variants. - Deoxyribonucleic acid (DNA) of all organisms is subjected to a wide range of mutagenic agents. Base deamination is a major type of DNA damage under nitrosative stress, but it can occur spontaneously as well, generating the base analogs, which have pairing specificities different from the original bases. Endonuclease V (EndoV) is a repair enzyme, which initiates removal of deaminated bases from damaged DNA. It is also called deoxyinosine 3 endonuclease, as it preferentially cleaves DNA containing deoxyinosine, a deamination product of deoxyadenosine. However, EndoV may also recognize deoxyxanthosine, deoxyoxanosine, deoxyuridine, abasic (AP) sites, base mismatches, flap DNA, pseudo-Y structures, and small insertions/deletions in DNA molecules [1, 2]. The cleavage site generated by EndoV occurs at the second phosphodiester bond in the 3 direction from the lesion, leaving a nick with 5-phosphate and 3-hydroxyl groups [1]. EndoV requires Mg2+ or Mn2+ ions for its activity. Although there is no general agreement on the number of metal ions involved in catalysis, recently a catalytic and regulatory two-metal model has been proposed, similar to the one proposed for restriction endonucleases [3]. According to this model, EndoV possesses two metal binding sites, M1 and M2. Occupation of the M1 site by a catalytic metal (Mg2+ or Mn2+) is required for catalysis and the M1 site has relatively high affinity for metal ions. Occupation of the M2 site is not essential for catalysis, but it can regulate the activity catalyzed by the metal ion located in the M1 site. The M2 site can be occupied by Mg2+ or Mn2+, as well as by Ca2+. On the other hand, if Ca2+ is located in the M1 site, it inhibits the cleavage reaction. Alternatively, EndoV may follow the mechanism proposed for RNase H, in which both metal ions are catalytic [4]. EndoV homologs have been found in Eubacteria, Archaea and Eukaryota. Prokaryotic members of the family are approximately 200 amino acids in length, while the mammalian homologs are about 100 aa longer due to the Cterminal extension. Some EndoV family proteins have additional domains leading to significant enlargement of the entire protein (e.g., C. elegans enzyme is 758 aa long). Sequence alignments of EndoV homologs allowed identifying seven conserved regions universal to all EndoV family proteins [5]. Motif I contains an invariant Gln residue that is moderately important for substrate and product binding. Motif II includes the active site Asp residue that is essential for catalysis. Motifs IIIVI contain many residues that are directly or indirectly involved in proteinDNA interactions [5]. Site-directed mutagenesis analysis of residues in conserved motifs revealed that D43 in motif II, E89 in motif III, and D110 in motif IV of T. maritima EndoV, hereafter referred to as TmEndoV are involved in metal cofactor coordination and catalytic function (in E. coli enzyme, hereafter referred to as EcEndoV, these residues correspond to D35, E82, D103, respectively). The fourth highly conserved residue, H214 in TmEndoV (D206 in EcEndoV), has been suggested to play a role in metal binding, nonetheless is the most tolerant to mutagenesis, e.g., it is exchangeable between Asp and His in the EndoV family [3]. Tyrosine at position 80 of TmEndoV (Y73 in EcEndoV) was shown to play a role in substrate and product binding, and to be important in the context of base preferences of mismatch cleavage [5]. Interestingly, substrate preference of EndoV homologs varies among different organisms. EcEndoV has a wide substrate spectrum, while EndoV from A. fulgidus and H. sapiens recognize only deoxyinosine [6]. It has been suggested that the deoxyinosine cleavage activity is a primordial activity of EndoV enzymes and that the ability of some bacterial members of this family to recognize other DNA lesions was acquired later during the course of evolution [7]. While TmEndoV can rapidly turn over T/Ucontaining double-stranded DNA [2], S. typhimurium EndoV can only turn over deoxyuridine-containing DNA to a limited extent when the substrates are in excess, likely due to tighter binding to these substrates [8]. For EcEndoV the mismatchspecific activity of the enzyme is reduced when the mismatch is flanked by GC pairs, while its deoxyinosine-specific activity is not influenced by the sequence context [9]. Nonetheless, amino acid residues essential for deaminated base recognition and DNA cleavage are highly conserved. The deoxyinosine or the damaged bases are not removed from DNA by EndoV, and the enzyme forms a stable complex with mutated DNA both before and after cleavage. Therefore, it has been proposed that, besides its endonuclease activity, the enzyme might function to target other repair protein(s), initiating a repair pathway [1]. It was also hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5 endonuclease or a 35 exonuclease to remove the damaged base and DNA polymerase and DNA ligase for repair action [10]. After discovery of EndoV 3-exonuclease activity, the alternative model has been proposed, in which EndoV plays a dual role in the repair process. According to this model, additional protein(s) may induce a conformational change in EndoV, causing switch from endonuclease to 3exonuclease mode, progressive removal of nucleotides from 3 side to the 5 side, and gap creation for repair synthesis [5]. Regrettably, no structural information is available for this interesting enzyme to (...truncated)