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

“Mirror” Method to Estimate Mutagenic Activity of DNA Lesions

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered

Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method

We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos

Alterations in Synthesis and Repair of DNA during the Development of Loach Misgurnus fossilis

Using a modified radiolabeled primer extension method (we named this modification misGvA—“misincorporation of G versus A”) we have investigated the DNA synthesis and repair at early and late stages of development of loach Misgurnus fossilis. The misincorporation activity of DNA polymerase iota (Pol ι) in wild-type loach could not be detected by this method at any stage of loach development. In transgenic loach overexpressing human Pol ι we have shown that the bypassing of DNA synthesis arrest after incorporation of mismatched nucleotide by Pol ι (the T-stop) was not associated with this enzyme. Non-transgenic loach larvae are virtually lacking the capacity for error correction of DNA duplex containing a mismatched nucleotide. Such repair activity develops only in the adult fish. It appears that the initial stages of development are characterized by more intensive DNA synthesis, while in terminal stages the repair activities become more prominent. The misGvA approach clearly indicates substantial changes in the DNA synthesis intensity, although the role of particular replicative and repair DNA polymerases in this process requires further study

Microcin C51 Plasmid Genes: Possible Source of Horizontal Gene Transfer

Microcin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural Escherichia coli strain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the mcc genes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The mccA, mccB, mccD, and mccE genes are involved in MccC51 production, while the mccC and mccE genes are responsible for immunity to MccC51. The mcc gene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned mccA, mccB, mccC, and mccE genes, but lacking mccD, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against E. coli and was toxic to the producing cells. The immunity to exogenous MccC51 determined by the mccC and mccE genes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the E. coli genome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster

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

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

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