Data on PAGE analysis and MD simulation for the interaction of endonuclease Apn1 from Saccharomyces cerevisiae with DNA substrates containing 5,6-dihydrouracyl and 2-aminopurine

This article presents new data on nucleotide incision repair (NIR) activity of apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae, which is known as a key player of the base excision DNA repair (BER) pathway, see “Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV” [1], “Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae” [2] and “Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases” [3]. The characterization of NIR activity of wild type Apn1 and mutant form Ape1 H83A were made by denaturing PAGE analysis, and MD simulations of Apn1 complexed with DNA containing 5,6-dihydro-2′-deoxyuridine (DHU) and 2-aminopurine (2-aPu) residues. This data article is associated to the manuscript titled “Apurinic/apyrimidinic endonuclease Apn1 from Saccharomyces cerevisiae is recruited to the nucleotide incision repair pathway: kinetic and structural features” [4]. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

apyrimidinic endonuclease Apn1 from Saccharomyces cerevisiae is recruited to the nucleotide incision repair pathway: kinetic and structural features" [4].
Published Value of the data The data of MD simulation provide information for the structures of WT Apn1 complexed with NIR substrates, containing 5,6-dihydrouracil and 2-aminopurine residues.
The data illustrates that efficiency of NIR catalysis driven by Apn1 depends strongly on the spatial structure of DNA-substrates.
The data could be useful guidelines for further design of new anti-fungal and anti-malarial agents as much as yeast Apn1 belongs to Endo IV family, which members are not found in mammalian cells, but are present in many microorganisms.

Data
Data reported here describe the features of nucleotide incision repair (NIR) of DNA catalyzed APendonuclease by Apn1 from Saccharomyces cerevisiae as revealed from kinetic studies and MD simulation analysis. To optimize the kinetic scheme, which would be describe the kinetic traces obtained by stoppedflow technique [4], the proposed mechanisms should be examined by adding a gradual stage of the enzyme-substrate complex transformation, with replot and analysis of residuals being carried out. Global nonlinear least-squares fitting of the data obtained was performed in the DynaFit software (BioKin Ltd., USA) [5]. The scree test was conducted for validation of the proposed kinetic scheme (Fig. 1). Two-or three-step binding mechanisms describing Apn1's interaction with substrate DHU(2-aPu) in BER buffer are represented as Schemes 1 and 2, respectively.

The influence of Mg2 þ concentration
Dependence of AP endonuclease activities of WT or H83A Apn1 on Mg 2 þ ion concentration was tested using 12mer DNA duplexes containing tetrahydrofuran analog of AP site (F), and downstream mispaired 2-aPu residue. The main difference of NIR and BER buffers is 5 mM Mg 2 þ ions presence or absence, respectively (Fig. 2).

Study of NIR activity of WT Apn1
NIR activity of WT Apn1 was recorded by stopped-flow technique [4] (2-aPu fluorescence intensity detection) or monitored using denaturing PAGE (Fig. 5).

Kinetic data analysis
Global nonlinear least-squares kinetic analysis was performed in the DynaFit software (BioKin Ltd., USA) [5] as described in [9,10].

An incision assay
The DNA cleavage kinetics in vitro conditions was studied using electrophoresis in polyacrylamide gel (PAGE) as described previously [6,7]. The measurements were conducted at 25°C in BER or NIR

MD simulations
The initial structure of a DNA duplex (PDB ID: 2NQJ [11]) was manually truncated to a 12mer and edited according to a nucleotide sequence being studied containing 2-aPu and/or DHU residues. Zn 2 þ ions were placed in the PDB file according to refs. [12,13] and the data obtained on the CheckMyMetal server and RaptorX-Binding server [14]. Parameterization of Zn 2 þ ions in a protein for MD simulations remains a challenge with classical mechanics. In this work, we tested different approaches to  Zn 2 þ parameterization: the cationic dummy atom (CaDA) approach [15] that involves virtual atoms to impose an orientational requirement for zinc ligands; the polarizable atomic multipole-based electrostatic model [16]; and the classic nonbonded atom method [17]. Finally, we found that the nonbonded atom method is more suitable for our purposes; accordingly, in this work, we chose this approach. Parameterization of Zn 2 þ ions was carried out as in ref. [17]. Structure refinement and molecular dynamic simulation were performed as in [7] using AMBER 14 molecular modeling suite [18,19]. The force field parameters for the 2-aminopurine-5 0 -phosphate residue were retrieved from ref. [20]. The partial atom charges and force fields for the DHU residue were custom-parameterized calculated by the RESP method [21] based on the quantum mechanical calculation in the HF/6-31 G* using Gaussian'09 software [22]. A 45 ns MD simulation was conducted using the AMBER 14 GPUaccelerated code [18,23] by means of the ff99SB force field [24,25].Molecular graphics, MD movie generation, and trajectory analysis were carried out in the UCSF Chimera software [26].