ITPA protects against HAP-induced DNA breaks.

<p>Alkaline comet assay data reveals that as compared to the control and non-targeting shRNA-expressing cell lines, ITPA knockdown cells accumulated elevated levels of DNA breaks after 24 hours of treatment with HAP. At high doses of HAP, the sizes of the comet tails in the ITPA knockdown cells were too large to be quantified. The assay was performed at lower doses of HAP treatment in order to obtain measurable comet tails. As compared to the cells with vector, the overexpression of <i>HAM1</i> suppressed the accumulation of HAP-induced DNA breaks in the <i>ITPA</i> knockdown cells. Statistical differences were measured by one-way ANOVA followed by Dunns multiple comparisons for column analysis. By two-way ANOVA, p = 0.008. Post-test analysis revealed differences (p<0.05) for knockdown versus knockdown+<i>HAM1</i> and knockdown+vector versus knockdown+<i>HAM1</i>. No difference in levels of DNA breaks was observed for hydrogen peroxide treatment for all three cell lines.</p

Model for the protective role of ITPA against HAP-induced genotoxicity and mutagenesis.

<p>In the presence of functional ITPA, the accumulation of non-canonical nucleotides like dHAPTP is abrogated by the ITPase, thereby preventing their incorporation into DNA. In the absence of functional ITPase, dHAPTP accumulates in the precursor pool and is incorporated into DNA by the replicative DNA polymerases. Grey circles represent HAP accumulation in DNA. Slow excision of base analogs by an unknown nuclease/glycosylase results in the accumulation of single-strand DNA breaks, which triggers apoptosis. Increased levels of apoptosis contribute to the onset of degenerative diseases. In the absence of repair, HAP persists in DNA causing incorrect pairing with T or C, thus leading to the accumulation of mutations, which predisposes individuals to the development of cancer.</p

ITPA protects against HAP-induced mutagenesis.

<p>The data represent the induced <i>HPRT</i> mutant frequency for the HAP-treated control and ITPA knockdown cells. At a low dose (0.1 mM) of HAP treatment for 24 hours the difference between cell lines was not significant but at dose 1 mM ITPA knockdown cells were more sensitive to HAP mutagenesis. (**p<0.01, n.s., not significant).</p

HAP-induced apoptosis occurs through the intrinsic pathway.

<p>(A) Protection from HAP-induced apoptosis by overexpression of Bcl-xL. Both HeLa and HeLa-xL cell lines were treated with increasing doses of HAP for 48 hours and the percentage of apoptotic cells was determined by Hoechst staining. By two-way ANOVA, p_{cell line}<0.0001 and p_{concentration} = 0.009. **p<0.01,***p<0.001 by Bonferroni multiple comparison post test for column analysis comparing means for HeLa vs. HeLa-xL hours. (B) Confirmation of protection from HAP-induced apoptosis by Bcl-xL overexpression by immunoblot for PARP cleavage. HAP induced dose-dependent cleavage of PARP in regular HeLa cells but not in HeLa cells overexpressing Bcl-xL. PAPR cleavage product is marked as cl₈₉. (C) HAP treatment results in similar levels of DNA breaks in HeLa and HeLa+Bcl-xL cell lines (p>0.05).</p

ITPase overexpression suppresses HAP-induced cytotoxicity.

<p>(A) Effect of overexpression of the <i>ITPA</i> and the gene encoding yeast ITPase, <i>HAM1</i>, on HAP-induced apoptosis in HeLa cells. Differences are significant (*p<0.05, **p<0.01). (B) Overexpression of the yeast <i>HAM1</i> could rescue <i>ITPA</i> knockdown cells from hypersensitivity to HAP-induced apoptosis (****p<0.0001, n.s., not significant).</p

HAP treatment leads to the appearance of EndoV sensitive sites in HeLa DNA.

<p>We extracted genomic DNA from HeLa cells grown with or without HAP. Treatment of this DNA with bacterial EndoV creates 3′ nicks, which are substrates for nick-translation (BioProbe® Nick translation kit with bio-16-dUTP (Enzo Life Sciences)) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032313#s4" target="_blank">Materials and Methods</a>. A. Agarose gel electrophoresis of nick-translated DNA from HeLa cells. 1- from untreated cells; 1a – from untreated cells digested with DNase; 2 – from cells grown in 2.64 mM HAP; 3- from untreated cells, DNA incubated with Endo V; and 4 - from cells grown in 2.64 mM HAP, DNA incubated with Endo V. B. Detection of newly synthesized biotinylated DNA separated by alkaline agarose electrophoresis. 1- from untreated cells; 2 – from cells grown in 2.64 mM HAP; 3- from untreated cells, DNA incubated with Endo V; and 4 - from cells grown in 2.64 mM HAP, DNA incubated with Endo V.</p

ITPA protects against HAP-induced apoptosis.

<p>ITPA knockdown sensitizes cells to HAP-induced apoptosis. As compared to the control and non-targeting shRNA transfected cells, ITPA knockdown cells undergo approximately 30–50% apoptosis upon HAP treatment for 24 hours. Hydrogen peroxide treatment (0.1 mM, four hours) was used as a positive control. The difference between control cells and cells with the <i>ITPA</i> knockdown is highly significant (***p<0.001, ****p<0.0001). There was no difference between the control versus the non-targeting cell lines in all HAP doses tested. No significant difference in hydrogen peroxide-induced apoptosis was observed for all three cell lines.</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

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes

<div><p>Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered <i>kataegis</i> in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced <i>can1</i> mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the <i>sub1</i> clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and <i>kataegis</i> events in different tumor cells.</p></div

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