10 research outputs found
Purification and the catalytic activity of PrimPol from different expression systems.
<p><b>(</b>A) purified preparation of GST-tagged PrimPol from different <i>E</i>. <i>coli</i> strains and <i>S</i>. <i>cerevisiae</i>. Coomassie staining. (B) the comparison of the DNA polymerase activity of PrimPol purified from different strains of <i>E</i>. <i>coli</i>. 75ā450 nM of GST-tagged PrimPol and 100 nM 30-mer DNA template were used in the reactions. (C) purified preparation of PrimPol from human FreeStyle 293-F cells. Coomassie staining. (D) the comparison of the DNA polymerase activity of PrimPol purified from ArcticExpress(DE3)-pRIL <i>E</i>. <i>coli</i> cells and human FreeStyle 293-F cells. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. Experiments (B and D) were repeated four times.</p
The stability of the PrimPolās catalytic activity <i>in vitro</i>.
<p>(A) the DNA polymerase activity of PrimPol after the indicated time pre-incubation on ice. (B) the DNA polymerase activity of PrimPol after the indicated amount of freeze-thaw cycles. (C) the DNA polymerase activity of PrimPol after 30 min incubation at 0ĀŗC, 25ĀŗC, 30ĀŗC, 35ĀŗC, 37ĀŗC and 40ĀŗC. (D) the DNA polymerase activity of PrimPol after incubation at 37ĀŗC. 450 nM of PrimPol and 100 nM 30-mer DNA template were used in the reactions. Experiments were repeated two times in A and B and three times in C and D.</p
The reaction buffer composition affects the DNA polymerase activity of PrimPol <i>in vitro</i>.
<p>(A) the DNA polymerase activity of PrimPol at different NaCl concentrations. (B) the DNA polymerase activity of PrimPol at different pH. HEPES based buffer was used to test the DNA polymerase activity of PrimPol at different pH values. (C) the DNA polymerase activity of PrimPol at different MgCl<sub>2</sub> and MnCl<sub>2</sub> concentrations. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. All experiments were repeated three times.</p
Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol - Table 1
<p>Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol</p> - Table
Bypass of a Psoralen DNA Interstrand Cross-Link by DNA Polymerases Ī², Ī¹, and Īŗ in Vitro
Repair of DNA interstrand cross-links in mammalian cells
involves
several biochemically distinctive processes, including the release
of one of the cross-linked strands and translesion DNA synthesis (TLS).
In this report, we investigated the in vitro TLS activity of a psoralen
DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases
Ī², Īŗ, and Ī¹. DNA polymerase Ī² is capable of
bypassing a psoralen cross-link with a low efficiency. Cell extracts
prepared from DNA polymerase Ī² knockout mouse embryonic fibroblasts
showed a reduced bypass activity of the psoralen cross-link, and purified
DNA polymerase Ī² restored the bypass activity. In addition,
DNA polymerase Ī¹ misincorporated thymine across the psoralen
cross-link and DNA polymerase Īŗ extended these mispaired primer
ends, suggesting that DNA polymerase Ī¹ may serve as an inserter
and DNA polymerase Īŗ may play a role as an extender in the repair
of psoralen DNA interstrand cross-links. The results demonstrated
here indicate that multiple DNA polymerases could participate in TLS
steps in mammalian DNA interstrand cross-link repair
Expression of the <i>hPOLISc</i> or <i>hPOLHSc</i> in yeast is non-mutagenic.
<p>* Median for 12-18 independent cultures. The 95% confidence limits are in parenthesis.</p
Activity of pure GST-tagged Pol Ī¹ variants.
<p>A. Purification of GST-Pol Ī¹ and its variants by affinity chromatography: the photograph of a Coomassie brilliant blue stained gel is shown. Equal volumes (15 Āµl) of each fraction with wild-type GST-Pol Ī¹, GST-Pol Ī¹<sup>D34A</sup>, GST-Pol Ī¹<sup>D126A/E127A</sup>, GST-Pol Ī¹<sup>D34A/126A/E127A</sup>, and GST-Pol Ī¹<sup>L62I</sup> eluted from the glutathione-sepharose column were analyzed on 8% SDS-PAGE. B. The comparative DNA-polymerase assay with purified GST-Pol Ī¹ and its variants. The ability of enzymes to extend a P<sup>32</sup>-labeled 17-mer primer annealed to template 1 was assayed in the presence of 100 ĀµM of all four dNTPs and 0.15 mM Mn<sup>2+</sup> ions, at 37Ā°C for 5 min. C. Kinetic analysis of dATP and dGTP incorporation by purified wild-type GSTāPol Ī¹ and GSTāPol Ī¹ <sup>L62I</sup> variant. Primer extension reaction was carried out in the presence of 0.15 mM Mn<sup>2+</sup> divalent metal ions and 1 nM of GST-Pol Ī¹ or its catalytically compromised variant at 37Ā°C for 2.5 min. To quantify the incorporation of dATP and dGTP opposite template T we varied each dNTP concentration from 0.3 to 100 ĀµM. Kinetic parameters determined from these experiments were: Wild-type: dATP: K<sub>m</sub>ā=ā3.5Ā±1 ĀµM, V<sub>max</sub>ā=ā9.8Ā±0.8 (% incorporation/min), dGTP: K<sub>m</sub>ā=ā0.57Ā±0.08 ĀµM, V<sub>max</sub>ā=ā14.9Ā±0.3 (% incorporation/min), f<sub>inc</sub> for dGTPā=ā5.3; and L62I: dATP: K<sub>m</sub>ā=ā4.0<0.9 ĀµM, V<sub>max</sub>ā=ā11.4Ā±0.8 (% incorporation/min), dGTP: K<sub>m</sub>ā=ā0.54Ā±0.09 ĀµM, V<sub>max</sub>ā=ā17.2Ā±0.3 (% incorporation/min), f<sub>inc</sub> for dGTPā=ā6.1.</p
misGvA by yeast extracts producing variants of Pol Ī¹.
<p>A. misGvA activity of cell extracts of yeasts producing GST-Pol Ī¹ variants was estimated by āmisGvAā in 8-min reactions in the presence of 0.15 mM Mn<sup>2+</sup> and 0.25 mM Mg<sup>2+</sup> divalent metal ions at template 1 as described in ā<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#s4" target="_blank">Materials and Methods</a>.ā Two combinations of equal concentrations of 1 mM dNTPs have been used: 1) all dNTPs and 2) dGTP and dATP. B. Same with template 2. B. Western-blot analysis of the production of GST-tagged Pol Ī¹ catalytically compromised variants in yeast cells extracts. Extracts containing 40 Āµg of total protein were separated on 8% polyacrylamide-SDS gel, transferred to a PVDF membrane and probed with polyclonal anti-GST antibodies. Cross-reacting proteins were visualized according to the Western Breeze Chromogenic Anti-Rabbit Kit procedure (Invitrogen).</p
Detection of misGvA activity of Pol Ī¹.
<p>A. DNA-polymerase activity of yeast extracts producing Pol Ī¹ or transformed by empty vector in the absence of dNTPs in reaction mixture. The reactions were carried out at 37Ā°C for 15 min as described in ā<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#s4" target="_blank">Material and Methods</a>ā using standard template 1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#pone.0016612-Zhang1" target="_blank">[4]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#pone.0016612-Gening2" target="_blank">[31]</a>, with no dNTPs in the reaction mixture. DNA oligonucleotides were separated on 18% polyacrylamide/7 M urea gels and visualized using Storage Phosphor Screen in Typhoon 9700. B. misGvA activity by yeast extracts producing human Pol Ī¹, Pol Ī· or transformed with empty vector. Template 1 (left panel, standard substrate) and template 2 (modified substrate with the substitution of C to G at the position of 19) were incubated with yeast cell extracts and high concentrations of exogenous nucleotides. The activity of Pol Ī¹ produced in yeast is detectable by the misincorporation of dGTP opposite template T by whole cell extracts (āmisincorporation of Gā method of Gening, abbreviated as misGvA). The cells extracts of yeast producing Pol Ī¹, Pol Ī· or containing empty vector were used as an enzymatic preparation for DNA polymerase reaction with P<sup>32</sup>-labeled oligonucleotide substrate in the presence of 0.15 mM Mn<sup>2+</sup> divalent metal ions and various combinations of equal concentrations of 1 mM dNTPs. For each extract, five different conditions have been used: 1) all dNTPs, 2) dATP, dTTP and dCTP but with dGTP omitted, 3) dGTP and dATP, 4) only dATP, and 5) only dGTP. Template 2 with the substitution of the next nucleotide upstream (+2, corresponding to the position of 19 on elongated primer) after the T template (+1, position of 18) from C to G was used to exclude the possibility of transient misincorporation by the template slippage mechanism. The reactions were carried out at 37Ā°C for 15 min. DNA products were separated on 18% polyacrylamide/7 M urea gels and visualized using Storage Phosphor Screen in Typhoon 9700. The Pol Ī¹ activity was determined by the presence of the two ādoubletā radioactive bands corresponding to 18-mer oligonucleotides with A or G at the 3ā²-end, possessing different electrophoretic mobility. The lower band with higher mobility is indicative of the amount of oligonucleotide with 3ā²-terminal A, whereas the upper band corresponds to the amount of a less mobile oligonucleotide with G incorporated opposite T in position 18 (lines 1 and 3).</p
Structure of Pol Ī¹ and the variants studied.
<p>A. Upper half. The schematic domain structure of Pol Ī¹ is shown (PIP is the protein interaction domain, UBM ā ubiqutin-binding motif). Lower half. The Pol Ī¹ mutant variants were used in the study (Red bars on the thick blue line representing Pol Ī¹ show the positions of amino acid changes in polymorphic variants of Pol Ī¹. Grey bars indicate deletions for truncated Pol Ī¹ variants): <i>hPOLISc<sup>2exon-d</sup></i> (Pol Ī¹ <sup>2exon-d</sup>) ā Pol Ī¹ variant with a deletion of exon 2 (encoding for amino acids 14-55) representing an alternative splice variant of human and mouse Pol Ī¹; <i>hPOLISc<sup>D34A</sup></i> (Pol Ī¹ <sup>D34A</sup>) and <i>hPOLISc<sup>D126A/E127A</sup></i> (Pol Ī¹ <sup>D126A/E127A</sup>) ā ācatalytically deadā Pol Ī¹ variants created as amino acid substitutions of evolutionary conservative Asp34 or Asp126 and Glu127 to Ala; <i>hPOLISc<sup>D34A/D126A/E127A</sup></i> (Pol Ī¹<sup> D34A/D126A/E127A</sup>) ā a triple ācatalytically deadā Pol Ī¹ variant with amino acid substitutions of Asp34, Asp126 and Glu127 to Ala; <i>hPOLISc<sup>L62I</sup></i> (Pol Ī¹<sup>L62I</sup>) ā Pol Ī¹ variant with a substitution of evolutionary polymorphic amino acid Leu62 to Ile; <i>hPOLISc<sup>42I-612-d</sup></i> variant with a deletion of the C-terminal half of Pol Ī¹ is shown to illustrate what minimal part retains enzymatic activity and whose crystal structure has been determined. B. PolĪ¹ active site. A close view at the Pol Ī¹ active site in ternary complex with DNA (template T) and incoming dGTP (3gv8). Pol Ī¹ and DNA molecules are represented as cartoon and sticks, respectively. The incorporated nucleotides and active site residues Asp34, Asp126 and Glu127 are represented by sticks and highlighted with yellow carbons. The side chains of phosphate-binding residues are also shown as sticks. The metal ion is drawn as a magenta ball.</p