Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs

Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer

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

Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication stress-induced fragile site breakage.

Chromosomal fragile sites are genomic loci sensitive to replication stress which accumulate high levels of DNA damage, and are frequently mutated in cancers. Fragile site damage is thought to arise from the aberrant repair of spontaneous replication stress, however successful fragile site repair cannot be calculated using existing techniques. Here, we report a new assay measuring recombination-mediated repair at endogenous genomic loci by combining a sister chromatid exchange (SCE) assay with fluorescent in situ hybridization (SCE-FISH). Using SCE-FISH, we find that endogenous and exogenous replication stress generated unrepaired breaks and SCEs at fragile sites. We also find that distinct sources of replication stress induce distinct patterns of breakage: ATR inhibition induces more breaks at early replicating fragile sites (ERFS), while ERFS and late-replicating common fragile sites (CFS) are equally fragile in response to aphidicolin. Furthermore, SCEs were suppressed at fragile sites near centromeres in response to replication stress, suggesting that genomic location influences DNA repair pathway choice. SCE-FISH also measured successful recombination in human primary lymphocytes, and identificed the proto-oncogene BCL2 as a replication stress-induced fragile site. These findings demonstrate that SCE-FISH frequency at fragile sites is a sensitive indicator of replication stress, and that large-scale genome organization influences DNA repair pathway choice

Human chromosome 1 satellite 3 DNA is decondensed, demethylated and transcribed in senescent cells and in A431 epithelial carcinoma cells

Constitutive heterochromatin mainly consists of different classes of satellite DNAs and is defined as a transcriptionally inactive chromatin that remains compact throughout the cell cycle. The aim of this work was to investigate the level of condensation, methylation and transcriptional status of centromeric (alphoid DNA) and pericentromeric satellites (human satellite 3, HS3) in tissues (lymphocytes, placenta cells) and in cultured primary (MRC5, VH-10, AT2Sp) and malignant (A431) cells. We found that alphoid DNA remained condensed and heavily methylated in all the cell types. The HS3 of chromosome 1 (HS3-1) but not of chromosome 9 (HS3-9) was strongly decondensed and demethylated in A431 cells. The same observation was made for aged embryonic lung (MRC5) and juvenile foreskin (VH-10) fibroblasts obtained at late passages (32nd and 23rd, respectively). Decondensation was also found in ataxia telangiectasia AT2Sp fibroblasts at the 16th passage. One of the manifestations of the disease is premature aging. The level of HS3-1 decondensation was higher in aged primary fibroblasts as compared to A431. The HS3-1 extended into the territory of neighbouring chromosomes. An RT-PCR product was detected in A431 and senescent MRC5 fibroblasts using primers specific for HS3-1. The RNA was polyadenylated and transcribed from the reverse chain. Our results demonstrate the involvement of satellite DNA in associations between human chromosomes and intermingling of chromosome territories. The invading satellite DNA can undergo decondensation to a certain level. This process is accompanied by demethylation and transcription

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

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