Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast., we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance

Activity of pure GST-tagged Pol ι variants.

<p>A. Purification of GST-Pol ι and its variants by affinity chromatography: the photograph of a Coomassie brilliant blue stained gel is shown. Equal volumes (15 µl) of each fraction with wild-type GST-Pol ι, GST-Pol ι^D34A, GST-Pol ι^D126A/E127A, GST-Pol ι^{D34A/126A/E127A}, and GST-Pol ι^L62I eluted from the glutathione-sepharose column were analyzed on 8% SDS-PAGE. B. The comparative DNA-polymerase assay with purified GST-Pol ι and its variants. The ability of enzymes to extend a P³²-labeled 17-mer primer annealed to template 1 was assayed in the presence of 100 µM of all four dNTPs and 0.15 mM Mn²⁺ ions, at 37°C for 5 min. C. Kinetic analysis of dATP and dGTP incorporation by purified wild-type GST–Pol ι and GST–Pol ι ^L62I variant. Primer extension reaction was carried out in the presence of 0.15 mM Mn²⁺ divalent metal ions and 1 nM of GST-Pol ι or its catalytically compromised variant at 37°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 µM. Kinetic parameters determined from these experiments were: Wild-type: dATP: K_m = 3.5±1 µM, V_max = 9.8±0.8 (% incorporation/min), dGTP: K_m = 0.57±0.08 µM, V_max = 14.9±0.3 (% incorporation/min), f_inc for dGTP = 5.3; and L62I: dATP: K_m = 4.0<0.9 µM, V_max = 11.4±0.8 (% incorporation/min), dGTP: K_m = 0.54±0.09 µM, V_max = 17.2±0.3 (% incorporation/min), f_inc for dGTP = 6.1.</p

Detection of misGvA activity of Pol ι.

<p>A. DNA-polymerase activity of yeast extracts producing Pol ι or transformed by empty vector in the absence of dNTPs in reaction mixture. The reactions were carried out at 37°C for 15 min as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#s4" target="_blank">Material and Methods</a>” 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 ι, Pol η 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 ι produced in yeast is detectable by the misincorporation of dGTP opposite template T by whole cell extracts (“misincorporation of G” method of Gening, abbreviated as misGvA). The cells extracts of yeast producing Pol ι, Pol η or containing empty vector were used as an enzymatic preparation for DNA polymerase reaction with P³²-labeled oligonucleotide substrate in the presence of 0.15 mM Mn²⁺ 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 37°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 ι activity was determined by the presence of the two “doublet” radioactive bands corresponding to 18-mer oligonucleotides with A or G at the 3′-end, possessing different electrophoretic mobility. The lower band with higher mobility is indicative of the amount of oligonucleotide with 3′-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

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

Structure of Pol ι and the variants studied.

<p>A. Upper half. The schematic domain structure of Pol ι is shown (PIP is the protein interaction domain, UBM – ubiqutin-binding motif). Lower half. The Pol ι mutant variants were used in the study (Red bars on the thick blue line representing Pol ι show the positions of amino acid changes in polymorphic variants of Pol ι. Grey bars indicate deletions for truncated Pol ι variants): <i>hPOLISc^2exon-d</i> (Pol ι ^2exon-d) – Pol ι variant with a deletion of exon 2 (encoding for amino acids 14-55) representing an alternative splice variant of human and mouse Pol ι; <i>hPOLISc^D34A</i> (Pol ι ^D34A) and <i>hPOLISc^D126A/E127A</i> (Pol ι ^D126A/E127A) – “catalytically dead” Pol ι variants created as amino acid substitutions of evolutionary conservative Asp34 or Asp126 and Glu127 to Ala; <i>hPOLISc^{D34A/D126A/E127A}</i> (Pol ι^{D34A/D126A/E127A}) – a triple “catalytically dead” Pol ι variant with amino acid substitutions of Asp34, Asp126 and Glu127 to Ala; <i>hPOLISc^L62I</i> (Pol ι^L62I) – Pol ι variant with a substitution of evolutionary polymorphic amino acid Leu62 to Ile; <i>hPOLISc^42I-612-d</i> variant with a deletion of the C-terminal half of Pol ι is shown to illustrate what minimal part retains enzymatic activity and whose crystal structure has been determined. B. Polι active site. A close view at the Pol ι active site in ternary complex with DNA (template T) and incoming dGTP (3gv8). Pol ι 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

misGvA by yeast extracts producing variants of Pol ι.

<p>A. misGvA activity of cell extracts of yeasts producing GST-Pol ι variants was estimated by “misGvA” in 8-min reactions in the presence of 0.15 mM Mn²⁺ and 0.25 mM Mg²⁺ divalent metal ions at template 1 as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016612#s4" target="_blank">Materials and Methods</a>.” 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 ι catalytically compromised variants in yeast cells extracts. Extracts containing 40 µg 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