MMS Treatment Research Articles

Opi1-mediated transcriptional modulation orchestrates genotoxic stress response in budding yeast.

In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.

Knockout of SlALKBH2 weakens the DNA damage repair ability of tomato

During the growth and evolution of plants, genomic DNA is subject to constant assault from endogenous and environmental DNA damage compounds, which will result in mutagenic or genotoxic covalent adducts. Whether for prokaryotes, eukaryotes or even viruses, maintaining genome integrity is critical for the continuation of life. Escherichia coli and mammals have evolved the AlkB family of Fe(II)/alpha-ketoglutarate-dependent dioxygenases that repair DNA alkylation damage. We identified a functional homologue with EsAlkB and HsALKBH2 in tomatoes, and named it SlALKBH2. In our study, the SlALKBH2 knockout mutant showed hypersensitivity to the DNA mutagen MMS and displayed more severe growth abnormalities than wild-type plants under mutagen treatment, such as slow growth, leaf deformation and early senescence. Additionally, genes with high transcriptional activity, such as rDNA, have increased methylation under MMS treatment. In conclusion, this study shows that the tomato SlALKBH2 gene may play an important role in ensuring the integrity of the genome.

Enhanced phosphorus mobility in a calcareous soil with organic amendments additions: Insights from a long term study with equal phosphorus input

The agricultural practice of replacing chemical fertilizers with organic amendments (manure and/or straw) may have consequences for phosphorus (P) loss to the environment. Such a knowledge gap was examined using a ten-year field trial in calcareous soil containing four treatments with the equal annual P input but varied organic amendment combinations as follows: mineral fertilizer only as control (MF), mineral fertilizer coupled with manure (MM), mineral fertilizer coupled with manure and straw (MMS) and mineral fertilizer coupled with straw (MS). The soil P distribution, P fractions and speciation, Fe(III) reduction and P sorption kinetics were investigated using the chemical extraction, K edge X-ray absorption near-edge structure and Langmuir equations. The electronic shuttle capacity of soils and speciation of soil dissolved organic matter (DOM) were also evaluated using electrochemical methods, three-dimensional excitation-emission matrix fluorescence spectroscopy and Fourier transform infrared spectra methods. Results showed that soil Olsen-P and total P increased at depths of 20–40 cm in MM, MMS and MS treatments, suggesting that manure and/or straw addition significantly mobilized P in the soil profile. Manure and/or straw addition also decreased soil maximum P sorption capacity (Smax) and increased the desorption rate at depths of 0–20 cm in soil across treatments. At a depth of 0–20 cm in soil of the MS treatment, the enhanced Fe(Ⅲ) reduction coupled with a decrease of Fe-bound P supports that Fe reduction dominates the mobilization of P. The transformation of Ca bound-P to Al/Fe bound-P in a depth of 0–20 cm in soil of the MM treatment may be due to the high proportion of humic-like substances in the DOM at a depth of 0–20 cm in soil of the MM treatment, which may have caused a slight/microsite acidification. These results can help to develop optimized fertilization practices to effectively mitigate P loss from calcareous soils with manure and/or straw addition.

Co-application of water management and foliar spraying silicon to reduce cadmium and arsenic uptake in rice: A two-year field experiment

Water management is an effective measure for the control of cadmium (Cd) and arsenic (As) in situ uptake and transport in rice. In this study, the effects of the co-application of foliar spraying silicon (Si) and water management on Cd and As uptake and transport in rice were studied under paddy soils that were seriously co-contaminated with Cd and As with a two-year field experiment. The results showed that the co-application of water management and foliar spraying Si could effectively decrease the bioavailability of Cd and As in soil and reduce the uptake and transport of Cd and As in rice. The co-application of water management and foliar spraying Si treatments decreased the exchangeable and TCLP extractable Cd and As contents in the soil. Especially for moisture at the maturing stage combined with foliar spraying Si treatment (MMS), the exchangeable and TCLP extractable Cd and As contents were significantly decreased by 48.49%–55.14% and 45.50%–54.67%, and 41.95%–56.73% and 37.80%–46.76% in the two seasons, respectively. The moisture at the maturing stage treatment significantly decreased the Cd and As contents in brown rice by 44.26%–48.59% and 23.90%–38.16% in the two seasons relative to the control, respectively. Furthermore, MMS treatment simultaneously inhibited Cd and As transport and accumulation in rice among all co-application treatments. The translocation factor (TF)stem-brown rice of Cd, TFstem-leaf of As, and TFstem-brown rice of As values in the MMS treatment were significantly decreased as compared with the MM treatment. Furthermore, both the Cd and As contents in brown rice under the MMS treatment significantly decreased by 15.33%–30.74% and 33.84%–40.80%, respectively, in the two seasons. The results suggested that foliar spraying Si combined with moisture at the maturing stage might be a promising measure to synchronously inhibit the transport and accumulation of Cd and As in rice.

Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA

BackgroundReversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations.MethodsHere we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry.ResultsMMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies.ConclusionsOur findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

Kleisin NSE4 of the SMC5/6 complex is necessary for DNA double strand break repair, but not for recovery from DNA damage in Physcomitrella (Physcomitrium patens)

Kleisin NSE4 and circular form of SMC5/6 is indispensable for DSB repair and necessary for gene targeting but is not enough for recovery of cells from DNA damage in Physcomitrella. Structural maintenance of chromosomes (SMC) complexes are involved in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play a prominent role in DNA stabilization during repair by tethering DNA at the site of lesion by a heteroduplex of SMC5 and SMC6 encircled with non-SMC components NSE1, NSE3 and kleisin NSE4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to DNA damage, kinetics of DSB repair and gene targeting. In the moss Physcomitrella (Physcomitrium patens), SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight reduction of transcript levels by dCas9 binding was enough to obtain stable lines with severe DSB repair defects and specific bleomycin sensitivity. We show that survival after bleomycin or MMS treatment fully depends on active SMC6, whereas attenuation of NSE4 has little or negligible effect. We conclude that circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the DSB repair, nevertheless there are other functions associated with the SMC5/6 complex, which are critical to survive DNA damage.

Characterization of chlorophyll deficient mutants in mungbean (Vigna radiata (L.) Wilczek)

A wide spectrum of chlorophyll mutants was obtained in M2 generation following mutagenesis with MMS, HZ and SA. All these chlorophyll deficient mutants were lethal except maculata, viridis and virescent. MMS treatments induced the highest frequency of chlorophyll mutations followed by HZ and SA. The frequency of chlorophyll mutations was dose dependent and increased with increasing concentrations of all the three mutagens utilized. Chlorina followed by xantha outnumbered the other types of chlorophyll mutants with all the three mutagens. Based on effectiveness, the order of mutagens was HZ > SA > MMS. Two criteria viz., pollen sterility (Mp/S) and seedling injury (Mp/I) were taken into consideration to determine the efficiency of the mutagens. With regard to these criteria, MMS mutagen was found to be the most efficient followed by HZ and SA. Lower concentrations of all the three mutagens were most effective and efficient in inducing chlorophyll mutations.

The HLTF\u2013PARP1 interaction in the progression and stability of damaged replication forks caused by methyl methanesulfonate

Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein–protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2′-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1–BARD1 interaction is further involved in the repair of collapse forks.

A 12-Week, Prospective, Non-Comparative, Non-Randomized Study of Magnetic Muscle Stimulation for Improvement of Body Satisfaction With the Abdomen and Buttocks.

Background and ObjectiveMagnetic muscle stimulation (MMS) is a relatively new energy‐based technology that provides a non‐invasive option for body contouring through stimulation and toning of underlying skeletal muscles. This study was conducted to examine the safety, efficacy, and body satisfaction scores of MMS using a CoolToneTM prototype for the aesthetic improvement of abdominal and buttock contour.Study Design/Materials and MethodsThis was a prospective, non‐comparative, non‐randomized, 12‐week, multicenter study. Male and female participants aged 22‐65 years received 4 MMS treatment sessions to the abdomen and/or buttocks. Body Satisfaction Questionnaire (BSQ) scores for abdomen and/or buttocks were assessed at baseline, immediately post final treatment, at 4 weeks (primary endpoint), and 12 weeks post final treatment. Subject‐rated Global Aesthetic Improvement Scale (SGAIS) was assessed at 4 weeks post final treatment (secondary endpoint), and 12 weeks post final treatment. Additional efficacy assessment included abdominal circumference obtained by 3D imaging at baseline, immediately post final treatment, and at 4 and 12 weeks post final treatment. A Subject Experience Questionnaire (SEQ) was used to assess treatment satisfaction and perspectives at 4 weeks and 12 weeks post final treatment. Adverse events (AEs) were monitored throughout the study.ResultsA total of 110 participants were recruited, who were 75% female, 80% Caucasian (mostly non‐Hispanic), average age of 39.5 years (range 22–59) with an average body mass index (BMI) of 23.3 kg/m2 (range 18–29.9). At the 4‐week post final treatment visit, the average BSQ score for participants receiving abdominal treatment (n = 93) was significantly improved with a 5.1 average increase in total score from baseline (possible score range 10–50) and by a 5.5 average increase from baseline for participants receiving buttocks treatment (n = 32) (p < 0.05). At 4 weeks post final treatment, the proportion of participants with SGAIS scores >“Improved” was 68.1% for participants receiving treatment of the abdomen (n = 94), and 81.8% for those receiving buttocks treatment (n = 33). The mean total decrease from baseline in waist circumference was significant at all time points. At the 12‐week post final treatment visit, SEQ data revealed that a majority of participants were “Satisfied” or “Very Satisfied” with overall treatment results and “Agreed” or “Strongly Agreed” that they were motivated to maintain results either by working out or by additional treatment. A total of 6 AEs related to the device and/or treatment were reported, which resolved spontaneously during the study.ConclusionTreatment of the abdomen and/or buttocks with MMS was well‐tolerated and demonstrated significant improvement in aesthetic appearance through the 12‐week post final treatment study duration. As a stand‐alone treatment, MMS expands the range of options for individualized treatment planning for patients seeking abdominal and/or gluteal muscle toning. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals, Inc.

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation.A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status.Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing.

Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans.

Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

Effects of dosage and durations of different mutagenic treatment in lentil (Lens culinaris Medik.) cultivars Pant L 406 and DPL 62

In order to broaden the genetic base of lentil, induced chemical mutagenesis was performed. Dry and healthy seeds of lentil cultivars Pant L 406 and DPL 62 were treated with 0.02% MMS (6hrs), 0.04% MMS (6hrs), 0.02% MMS (9hrs) and 0.04% MMS (9hrs) prepared in sodium phosphate buffer at 7.0 pH for 6 hours at room temperature. Bio-physiological observations showed almost a direct relationship with the MMS concentrations and treatment duration, where inhibition and sterility increases with increase in concentration and duration. The 0.02% and 0.04% for 6 hrs treatments of MMS were found effective in inducing sufficient genetic variability in both the cultivars of lentil especially for selection of yield attributing traits, and 0.04% for 9 hrs induced higher desirable phenotypic variations with few unique phenotypes of future interest, whereas 0.04% for 9 hrs found to have highest lethal dose value and % variations in the all variant small survival population. Plant habit and leaf morphology were found most sensitive phenotypic category towards the MMS doses used. Also the character association between yield and other agronomic traits were altered and improved due to the mutagenic treatments. Overall, the results were promising and useful for future crop improvement work on lentil.

Human sirtuin 3 (SIRT3) deacetylates histone H3 lysine 56 to promote nonhomologous end joining repair

Human sirtuin 3 (SIRT3) is a conserved NAD+ dependent deacetylase, which functions in important cellular processes including transcription, metabolism, oxidative stress response. It is a robust mitochondrial deacetylase; however, few studies have indicated its nuclear functions. Here we report interaction of SIRT3 with core histones and identified acetylated histone H3 lysine 56 (H3K56ac) as its novel substrate, in addition to known substrates acetylated H4K16 and H3K9. Further, we showed in response to DNA damage SIRT3 localizes to the repair foci colocalizing with γH2AX and nonhomologous end joining (NHEJ) marker p53-binding protein 1 (53BP1). However, it does not colocalize with homologous repair (HR) marker BRCA1. By ChIP break assay, we demonstrated the recruitment of SIRT3 at the double strand-break site in response to DNA damage. Additionally, the relocalization of SIRT3 to the nucleus on MMS treatment led to concurrent decrease in H3K56ac, which is an important step in NHEJ. Depletion of SIRT3 by si-RNA mediated knock down affected recruitment of 53BP1, resulting in compromised NHEJ efficiency, and survival defect as seen by colony formation assay. Altogether, our results demonstrated that SIRT3 recruits 53BP1 to the site of damage thereby plays a significant role in NHEJ pathway.

Statistical optimization for enhanced yields of probiotic Bacillus coagulans and its phage resistant mutants followed by kinetic modelling of the process.

Probiotics are microorganisms which when administered in adequate amounts confer health benefits to the host. A leading pharmaceutical company producing Bacillus coagulans as a probiotic was facing the problem of recurring phage attacks. Two mutants viz. B. co PIII and B. co MIII that were isolated as phage resistant mutants after UV irradiation and MMS treatment of phage sensitive B. coagulans parental culture were characterized at functional and molecular level and were noted to have undergone interesting genetic changes. The non-specific genetic alterations induced by mutagenesis can also lead to alterations in cell performance. Hence, in the current study the parental strain and the two mutants were selected for shake flask optimization. Plackett–Burman design was used to select the significant culture variables affecting biomass production. Evolutionary operation method was applied for further optimization. The study showed wide variations in the nutritional requirements of phage resistant mutants, post exposure to mutagens. An increment of 150, 134 and 152 % was observed in the biomass productions of B. coagulans (parental type) and mutants B.co PIII and B.co MIII respectively, compared to the yield from one-factor-at-a-time technique. Using Logistic and modified Leudeking–Piret equations, biomass accumulation and substrate utilization efficiency of the bioprocess were determined. The experimental data was in agreement with the results predicted by statistical analysis and modelling. The developed model may be useful for controlling the growth and substrate consumption kinetics in large scale fermentation using B. coagulans.

Non-Smc element 5 (Nse5) of the Smc5/6 complex interacts with SUMO pathway components.

ABSTRACTThe Smc5/6 complex in Saccharomyces cerevisiae contains six essential non-Smc elements, Nse1-6. With the exception of Nse2 (also known as Mms21), which is an E3 small ubiquitin-like modifier (SUMO) ligase, very little is understood about the role of these components or their contribution to Smc5/6 functionality. Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway. Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO. SUMO also mediates the interactions between Nse5 and the two PIAS family E3 SUMO ligases, Siz1 and Siz2. Cells carrying the nse5-ts1 allele or lacking either SIZ1 or SIZ2 exhibit a reduction in Smc5 sumoylation upon MMS treatment and demonstrate functional redundancy for SUMO mediated events in the presence of DNA damage. Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.

Differential response of biochemical parameters to EMS and MMS treatments and their dose effect relationship on chromosomes in induced diabetic mouse

AimTo study the effect of alkylating agents such as EMS and MMS on chromosomes and biochemical parameters in induced diabetic mouse. MethodsChromosome preparations from bone marrow was made using the method of Evans et al. (1964) and biochemical estimations from the liver was done by the method of Sinha (1972) for catalase, Van der Vies (1954) for glycogen and Uchiyama and Mihara (1978) for MDA. ResultsThe study has revealed that EMS and MMS induced a dose dependent increase in chromosomal aberrations of chromatid type in the diabetic mouse. Nonetheless, it is interesting to note that, there is significant reduction in the frequency of chromosomal aberrations in diabetic compared to non diabetic mice at all tested doses of EMS or MMS and at different recovery times [RTs]. On the other hand biochemical parameters showed a variable degree of reactivity: (1) catalase activity was significantly elevated in non diabetics whereas in diabetics it is significantly decreased with increasing concentrations of EMS. Contrary to this, the catalase activity in the case of MMS treatment is significantly reduced in non diabetics compared to diabetic mice. (2) However glycogen level is significantly reduced in both the diabetic and non diabetic with increasing concentrations of EMS or MMS, but MDA levels were significantly increased. Conclusion(1) Even though alkylating agents induce chromosomal aberrations in diabetic mice, MMS, a methylating agent is a more potent inducer of chromatid type of aberrations than EMS, an ethylating agent. (2) Diabetic mouse is more resistant than the non diabetic to alkylating agents and (3) the tested agents altered the analyzed biochemical parameters.

DiffNet: Automatic differential functional summarization of dE-MAP networks

The study of genetic interaction networks that respond to changing conditions is an emerging research problem. Recently, Bandyopadhyay et al. (2010) proposed a technique to construct a differential network (dE-MAPnetwork) from two static gene interaction networks in order to map the interaction differences between them under environment or condition change (e.g., DNA-damaging agent). This differential network is then manually analyzed to conclude that DNA repair is differentially effected by the condition change. Unfortunately, manual construction of differential functional summary from a dE-MAP network that summarizes all pertinent functional responses is time-consuming, laborious and error-prone, impeding large-scale analysis on it. To this end, we propose DiffNet, a novel data-driven algorithm that leverages Gene Ontology (go) annotations to automatically summarize a dE-MAP network to obtain a high-level map of functional responses due to condition change.We tested DiffNet on the dynamic interaction networks following MMS treatment and demonstrated the superiority of our approach in generating differential functional summaries compared to state-of-the-art graph clustering methods. We studied the effects of parameters in DiffNet in controlling the quality of the summary. We also performed a case study that illustrates its utility.

Analysis of Poly(ADP-Ribose) Polymerases in Arabidopsis Telomere Biology

Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one.

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.