Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., <i>N</i>²-ethyl­(Et)­G, <i>O</i>⁶-methyl­(Me)­G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, <i>N</i>²-EtG, <i>O</i>⁶-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the <i>N</i>-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg²⁺ (but not with Mn²⁺), coinciding with its steady-state kinetic data showing a ∼10-fold increase in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates (only with Mg²⁺). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates either with Mg²⁺ or Mn²⁺, except for that opposite <i>N</i>²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg²⁺), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn²⁺ than with Mg²⁺. The results indicate that the R96G variation severely impairs most of the Mg²⁺- and Mn²⁺-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg²⁺-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of <em>Taenia solium</em> Metacestode

<div><h3>Background</h3><p>Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of <em>Taenia solium</em> metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive.</p> <h3>Methodology/Principal Findings</h3><p>We isolated two novel <em>TsMFABP</em> genes (<em>TsMFABP1</em> and <em>TsMFABP2</em>), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, <em>cis</em>-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol.</p> <h3>Conclusions/Significance</h3><p>The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate <em>de novo</em> FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.</p> </div

Additional file 2: Figure S2. of Fasciclin-calcareous corpuscle binary complex mediated protein-protein interactions in Taenia solium metacestode

Efficiency of rTsMFas1 and rTsMFas2 in cellular adhesion. a Expression and purification of recombinant proteins. Bacterially expressed rTsMFas1 and rTsMFas2 proteins were purified using Ni-NTA column, after which His-tag was removed by thrombin cleavage. Proteins (each 200 ng) were monitored with 8% reducing SDS-PAGE followed by CBB staining. b Each well of a 96-well plate was coated with BSA (2 μg/ml), fibronectin (10 μg/ml) and each recombinant protein (10 μg/ml), after which incubated with MRC-5 and NHLF cells. Attached cells were measured by the hexosamidase assay. Graphic values of average and error bars representing standard deviations were obtained from triplicate assays of three independent experiments. (TIFF 220 kb

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

Expression patterns of TsMFABPs.

<p>(<b>A</b>) Proteins (each of 5 µg) extracted from the whole TsM (WE), bladder wall (BW), scolex and neck (SN), and cyst fluid (CF) were separated by 15% SDS-PAGE under reducing conditions, after which processed with immunoblotting probed with the respective antibodies or a preimmune mouse serum (1∶2000 dilutions). Proteins contained in the excretory-secretory products (ESP) were also included. The blots were developed with an ECL system. <i>M</i>_r, molecular mass in kDa. (<b>B</b>) Tissue locality of TsMFABPs was examined in the TsM sections by immunohistochemical staining. The whole image of evaginated worm stained with hematoxylin-eosin is shown (a). Worm sections were incubated with mouse antibodies specific to rTsMFABPs or a preimmune serum (1∶200 dilutions) and then, with rhodamine-conjugated anti-mouse IgG antibody. The slides were counterstained with DAPI (10 µg/ml). CA, spiral canal; FS, fibrillar stroma of BW; HT, hooklet; LT, loose tissue; NE, neck; NL, nuclear layer of BW; SC, scolex; SU, sucker.</p

Expression of mRNA transcripts of <i>TsMFABP</i> genes.

<p>Fresh TsM sections were probed with Cy5-labeled, <i>TsMFABP</i>-specific antisense or sense probes, after which counterstained with DAPI (10 µg/ml). Positive signals for <i>TsMFABP</i> expressions are shown in bright-pinkish color. BW, bladder wall; CA, spiral canal; CF, cyst fluid; NE, neck.</p

<i>In vitro</i> binding activity of native and recombinant TsMFABPs against DAUDA and retinol.

<p>Fluorescence emission spectra of DAUDA (0.1 µM, Ex_max = 345 nm) (<b>A</b> and <b>B</b>) and retinol (5 mM, Ex_max = 350 nm) (<b>C</b> and <b>D</b>) bound to the purified native (nTsMFABPs) (<b>A</b> and <b>C</b>) and recombinant (rTsMFABPs) (<b>B</b> and <b>D</b>) proteins (each 1 µM) were recorded at 25°C (200 µl/well) using a black 96-well micro-Fluor plate. DAUDA (10 mM stock dissolved in ethanol) was stored −20°C in darkness and were freshly diluted in ethanol just before use. The competitive binding of oleic acid (OA, 5 µM) to the performed rTsMFABP1:DAUDA or rTsMFABP2:retinol complex is also shown (<b>B</b> and <b>D</b>). TsM 120-kDa (TsM120 kDa, 10 µM) and recombinant 18-kDa (r18 kDa, 5 µM) proteins were included in the assay as negative controls.</p

Comparison of kinetic parameters for the binding reaction of the rTsMFARs against DAUDA, retinol and <i>cis</i>-parinaric acid (cPnA).

a<p>The rate was calculated by measuring relative fluorescence of each reaction.</p

Molecular properties of TsMFABPs.

<p>(<b>A</b>) The primary structures of TsMFABP1 and TsMFABP2 s were compared with those of related members. Dots represent gaps introduced into the sequences to increase similarity values. The secondary structure predicted with the TsMFABP1 sequence is shown at the top of the alignment. Brown boxes denoted as BC and FG loops indicate amino acid (aa) extensions found uniquely in TsMFABP2. Motifs 1, 2 and 3 are marked by blue boxes. The GXW triplet domain in motif 1 is shown by orange box. The aa residues involved in nuclear localization signal and its regulation site are shown by red and dotted red boxes, respectively. The hormone-sensitive lipase binding site is indicated by a blue arrow. Residues comprising the nuclear export signal are denoted by green boxes. Purple boxes indicate the putative signal sequences targeted for the phosphorylation. Red vertical line marked with a red arrow represents the position of orthologous introns conserved among the related proteins. TsMFABP1, HQ259679; TsMFABP2, HQ259680; <i>E. granulosus</i> FABP1 (EgFABP1), AAK00579; EgFABP2, AAK12095; <i>M. vogae</i> FABP (MvFABP), ABO93626; <i>Schistosoma japonicum</i> FABP (SjFABP), CAX71489; human muscle FABP (HsFABP_M), CAA39889; human brain FABP (HsFABP_B), NP_001437; human adipocyte FABP (HsFABP_A), NP_001433. (<b>B</b>) Phylogenetic positions of TsMFABPs and their platyhelminth orthologs were predicted among the human intracellular lipid binding family members such as FABP (_E, epidermis; _H, heart; _I, intestine; _L, liver; _Ila/b, ileal isoform a and b; mP2, myelin P2) and cellular retinol-/retinoic acid-binding protein (CRBP/CRABP). The tree was constructed using the neighbor-joining algorithm of MEGA and rooted with human plasma retinol binding protein (HsRBP). The statistical significance of the branching nodes (numerical in each of the nodes) was estimated by a bootstrapping analysis of 1000 replicates of initial input alignment. An internal node connecting major cestode proteins is indicated by a red arrow. CsFABP, <i>Clonorchis sinensis</i> FABP. (<b>C</b>) The genomic structures of <i>TsMFABP1</i> and <i>TsMFABP2</i> were determined from chromosomal segments homologous to each of the respective cDNAs. The lengths of predicted exons (rectangles) and introns (solid lines) are shown in base pairs. The open rectangles indicated the 5′- and 3′-untranslated regions. The initiation (ATG) and stop (TAA/TGA) codons in the open reading frame, as well as the nucleotides found in the exon-intron boundary (gt-ag), are marked in the corresponding positions. The intron phases are shown in parentheses.</p

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens