Figure 1.
Phylogenetic analysis and structural modeling of E. histolytica family A DNA Polymerase.
(A) Phylogenetic relationship of family A polymerases from different organisms. ClustalW multiple sequence alignment of diverse DNA polymerases were used to construct a phylogenetic tree using the neighbor joining algorithm present in the MEGA program. GenBank accession numbers of each protein are indicated in Table S2. The significance of each branch of the phylogenetic tree is indicated by a bootstrap percentage (B) Domain organization of EhDNApolA. EhDNApolA lacks the 3′-5′ exonuclease domain present in Klenow fragment. The polymerization domain of EhDNApolA is organized into three subdomains: thumb, palm, and fingers (C) Structural model of EhDNApolA. The N-terminal domain is shown in orange. The polymerization subdomains: thumb, palm, and fingers are colored in green, red, and cyan respectively. The model was constructed using the crystal structure of Klenow Fragment as a template (PDB ID: 1KFS).
Figure 2.
Heterologous expression and purification of E. histolytica family A DNA polymerase.
(A) Coomassie blue stained SDS-PAGE (10%) gel showing the expression and purification of EhDNApolA. Lane1, uninduced pCOLD-EhDNApolA construct; lane 2, IPTG induced sample; lane 3, insoluble fraction; lane 4, soluble lysate; lane 5, nickel agarose flow-through; lane 6, 35 mM imidazol wash; lane 7, 50mM imidazol wash; lane 8, nickel agarose column eluate; lane 9, phosphocelulose column eluate; lane 10, molecular weight standards. (B) Detection of recombinant and endogenous EhDNApolA.Recombinant EhDNApolA and total extracts of E. histolytica were resolved by SDS-PAGE (15%), electroblotted onto nitrocellulose membrane, and immunoblotted with diverse antibodies. Lane 1, recombinant EhDNApolA treated with commercial anti-6 histidines antibody; lane 2, recombinant EhDNApolA treated with mouse antibodies raised against an epitope of EhDNApolA; lane 3, total protein extracts of E. histolytica treated with preimmune serum; lane 4, total protein extracts of E. histolytica treated with specific mouse antibodies raised an epitope of EhDNApolA.
Figure 3.
Biochemical characterization of EhDNApolA.
(A) EhDNApolA binds to double stranded DNA. Increasing concentrations of recombinant EhDNApolA (from 0 to 180 nM) were incubated with a fixed amount of double stranded primer-template. The binding of the EhDNApolA to the primer-template is observed by the formation of a slower migrating complex on a native 6% polyacryalmide gel. (B) EhDNApolA is an active DNA polymerase. DNA polymerase activity was measured by the extension of 24mer primer annealed to a 45mer template. Lanes 1 and 4 contained reactions with no added polymerase, EhDNApolA (lanes 2 and 3) or Kf (exo-) (lanes 5 and 6). Reactions in lanes 2 and 5 were incubated with dTTP,dGTP and ddATP. Reactions in lanes 3 and 6 were incubated with all four dNTPs. Incorporation of ddATP results in extension to a 36mer and incubation with all four dNTPs results in a 45mer product. (C) Strand displacement activity of EhDNApolA. Strand displacement was determined using a six-nucleotide substrate gap depicted below Figure3D. The reactions contained 25 units of φ 29 DNA polymerase (lane 2), 4 units of Taq DNA polymerase (lane 3), 5 units of T7 DNA polymerase (lane 4), and 45 fmol of EhDNApolA (lane 5). The 18mer primer can be freely extended to a 24mer. Further extension is only possible if the DNA polymerase displaced the annealed 21mer.
Figure 4.
Nucleotide insertion fidelity of EhDNApolA.
16% denaturing polyacriylamide gel showing a primer-template extension by EhDNApolA in the presence of 100 µM of the indicated nucleotide. The first templated base is denoted with an X as depicted in figures A, B, C, and D. The identity of each dNTP in each reaction is indicated. The 24mer substrates and the 25mer products are indicated by an arrow. (A) Nucleotide fidelity for templated adenine (lanes 1 to 4). (B) Nucleotide fidelity for templated thymine (lanes 5 to 8). (C) Nucleotide fidelity for templated cytidine (lanes 9 to 12). (D) Nucleotide fidelity for templated guanine (lanes 13 to 16).
Figure 5.
Translesion DNA synthesis by EhDNApolA.
16% denaturing polyacrylamide gel electrophoresis showing translesion bypass of EhDNApolA. The first templated base is denoted with an X. The structure of each templated lesion and a templated thymine are depicted for comparison. The reactions were incubated with increased amounts of EhDNApolA (0, 60, 120, and 240 fmol) and 20pM of each substrate. Thymine (lanes 1–4); 8-oxo guanosine (lanes 5–8,); 5 S-6R thymine glycol (lanes 9–12); 5R-6S thymine glycol (lanes 13–16,); cis-syn cyclobutane pyrimidine dimer (lanes 17–20); 6-4 photo product (lanes 21–24); abasic site (lanes 25–29). The bottom arrow indicates the substrate length and the top arrow indicates the expected full-length products.
Figure 6.
Fidelity of translesion DNA synthesis by EhDNApolA.
16% denaturing polyacrylamide gel electrophoresis showing translesion bypass fidelity of EhDNApolA. 0.2 pmol of EhDNApolA were incubated with a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. (A) 8-oxo guanosine (lanes 1 to 6). (B) abasic site (lanes 1 to 6). (C) 5 S-6R thymine glycol (lanes 1 to 6). (D) 5R-6S thymine glycol (lanes 1 to 6). The identity of each nucleotide is indicated in the figure. The length of the substrates, single nucleotide extensions and full-length products are indicated by arrows.
Figure 7.
Cellular identification and localization of EhDNApolA.
(A) RT-PCR using total RNA from E. histolytica trophozoites grown in basal culture conditions 1% Agarose gel stained with ethidium bromide showing the RT-PCR products of motifs A (lane 3) and C (lane 4) of the EhDNApolA gene. The RT-PCR product of the actin gene control is shown in lane 2. (B) Immunodetection of EhDNApolA. Western blot assays using, cytoplasmic extracts (lane 1), and nuclear extracts (lane 2) of E. histolytica's trozophoites against mouse polyclonal anti- EhDNApolA antibodies (upper pannel). Control using mouse polyclonal anti-actin antibodies (middle panel) and anti- C/EBPβ antibodies (lower panel) (C–F) Cellular localization of EhDNApolA analyzed by confocal immunofluorescence microscopy. (C) Nomarsky image of a single E. histolytica cell stained with 4′,6-diamidino-2-phenylindole (DAPI) and incubated with anti-EhDNApolA antibodies treated with FITC-labeled secondary antibodies (D) fluorescence signal generated by DAPI (E) fluorescence signal generated by the binding of the FITC-conjugated antibody to anti-EhDNApol A antibody (F) Merge image of the fluorescence emitted by DAPI and FITC.