Methyl Methanesulfonate Treatment Research Articles

Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts

Alkylating agents modify DNA and RNA forming adducts that disrupt replication and transcription, trigger cell cycle checkpoints and/or initiate apoptosis. If left unrepaired, some of the damage can be cytotoxic and/or mutagenic. In Escherichia coli, the alkylation repair protein B (AlkB) provides one form of resistance to alkylating agents by eliminating mainly 1-methyladenine and 3-methylcytosine, thereby increasing survival and preventing mutation. To examine the biological role of the mammalian AlkB homologs Alkbh2 and Alkbh3, which both have similar enzymatic activities to that of AlkB, we evaluated the survival and mutagenesis of primary Big Blue mouse embryonic fibroblasts (MEFs) that had targeted deletions in the Alkbh2 or Alkbh3 genes. Both Alkbh2- and Alkbh3-deficient MEFs were ∼2-fold more sensitive to methyl methanesulfonate (MMS) induced cytotoxicity compared to the wild type control cells. Spontaneous mutant frequencies were similar for the wild type, Alkbh2−/− and Alkbh3−/− MEFs (average – 1.3×10−5). However, despite the similar survival of the two mutant MEFs after MMS treatment, only the Alkbh2-deficient MEFs showed a statistically significant increase in mutant frequency compared to wild type MEFs after MMS treatment. Therefore, although both Alkbh2 and Alkbh3 can protect against MMS-induced cell death, only Alkbh2 shows statistically significant protection of MEF DNA against mutations following treatment with this exogenous methylating agent.

ATMIN: A New Tumor Suppressor in Developing B Cells

In the current issue of Cancer Cell , Loizou et al. report that the deletion of ATM Interactor (ATMIN) in developing B cells leads to profound genomic instability and clonal B cell leukemia, revealing a novel tumor suppressor function for ATMIN.

Prooxidants prevent yeast cell death induced by genotoxic stress

It was shown earlier that DNA damage induced by alkylating agent MMS (methyl methanesulfonate) results in formation of ROS (reactive oxygen species) in yeast cells. Here, we asked whether this ROS generation is favourable for the cells. It appeared that prooxidants rather than antioxidants stimulate the survival after MMS treatment. We found that positively charged detergents increase the survival via induction of H2O2 formation in the cells. Interestingly, prooxidants protected yeast cells from the moderate doses of MMS and enhanced the toxicity of relatively high ones. Prooxidants also protect the cells arrested in mitosis (nocodazole treatment), indicating that the protection is mostly due to ROS-mediated transcriptional stress-response rather than due to enrichment of cell culture with highly MMS-resistant G2/M cells. The comparison of the published expression profile responses to prooxidant and MMS treatments identifies a set of ROS-activated genes, which are likely to protect cells from the genotoxic stress.

Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the SN2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PKCS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following SN2-alkylating agents.

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O 6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl- N′-nitro- N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

Sensitization to Radiation and Alkylating Agents by Inhibitors of Poly(ADP-ribose) Polymerase Is Enhanced in Cells Deficient in DNA Double-Strand Break Repair

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Abstract 2987: Oncogenic Wip1 is activated and negatively regulates DNA-damage induced apoptosis in p53-null cancer cells

Abstract Wip1 (wild-type p53-induced phosphatase, or PPM1D) belongs to the protein phosphatase type 2C family and its expression is induced by p53 in response to various stresses. Wip1 is known to dephosphorylate cellular target proteins such as p38 MAPK, p53, and uracil DNA glycosylase that are involved in DNA-repair and cell-cycle-checkpoint pathways. Here, we show that after methylmethane sulfonate (MMS) treatment Wip1 is induced in various breast-cancer cell lines in the absence of wild-type p53 and p38 MAPK activity. Wip1 expression in response to MMS was not blocked by the p38 MAPK inhibitor SB202190 or in p38-silenced MCF-7 cells using p38 MAPK small-interfering RNA (siRNA). The Wip1 promoter displayed great induction in breast cancer cell lines, which have DNA-binding deficient p53 mutants, and p53-null cancer cells such as Saos-2, A549-E6 and HCT116(p53−/−), whereas a control promoter, p53-Luc, showed the least promoter activity after MMS treatment. The mutation of p53 response element in the Wip1 promoter decreased its activity in both unstressed and stressed cells, but the overall level of fold-induction was similar. In addition, cell death was greatly increased in Wip1-siRNA and MMS treated cells in comparison with scrambled siRNA and MMS treated cells in the both cell lines of HCT116 (p53 +/+) and HCT116 (p53−/−). Our data indicate that Wip1 induction in response to various stimuli is not solely controlled by p53, and other damage response signaling appears to compensate for p53. Furthermore, Wip1 negatively regulates DNA-damage induced cell death regardless of the presence wild-type p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2987.

A Proteome-wide Analysis of Kinase-Substrate Network in the DNA Damage Response

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

Effects of Saccharomyces cerevisiae mec1, tel1, and mre11 mutations on spontaneous and methylmethane sulfonate-induced genome instability

In eukaryotes, together with the Mre11/Rad50/Xrs2 (or Nbs1) complex, a family of related protein kinases (the ATM family) is involved in checkpoint activation in response to DNA double-strand breaks. In Saccharomyces cerevisiae, two members of this family, MEC1 and TEL1, have functionally redundant roles in DNA damage repair. Strains with mutations in their mec1 as well as mre11 genes are very sensitive to DNA damaging agents, show defective induction of damage-induced cell-cycle checkpoints, and defective damage-induced homologous recombination. However, the fact that both the mec1Delta and mre11Delta strains exhibit the spontaneous hyper-recombination phenotype is paradoxical in light of the homologous recombination defects in these strains. In this study, we constructed yeast mec1, tel1, and mre11 null mutations and characterized their genome stability properties. Spontaneous and methylmethane sulfonate (MMS)-induced point mutations, base-substitutions, and frameshifts occurred to an almost equal extent in the wild-type, mec1Delta, tel1Delta, and mre11Delta strains. Thus, Mec1, Tel1, and Mre11 do not play roles in the point mutation response. We then found that the mec1Delta, mre11Delta, and mec1Delta tel1Delta strains demonstrated increased rates of spontaneous loss of heterozygosity (LOH), which includes crossover, gene conversion, and chromosome loss, compared with the wild-type strain. In the tel1Delta strain, the rate of spontaneous LOH was as low as that in the wild-type strain. Finally, no induction of LOH by MMS was observed in the mec1Delta, mre11Delta, or mec1Delta tel1Delta strain; however, it was detected in the wild-type and tel1Delta strains upon exposure to MMS. The elevated level of spontaneous LOH but not MMS-induced LOH in the mec1Delta, mre11Delta, and mec1Delta tel1Delta strains suggests the presence of high levels of spontaneous recombinogenic DNA damage, which differs from the damage induced by MMS treatment, in these strains.

Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2alpha, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2alpha kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2alpha kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2alpha phosphorylation. Finally, the MAPK Sty1, but not the eIF2alpha kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2alpha kinase pathways for a particular range of stress responses.

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.

Transcriptome and proteome analyses of adaptive responses to methyl methanesulfonate in Escherichia coli K-12 and ada mutant strains

BackgroundThe Ada-dependent adaptive response system in Escherichia coli is important for increasing resistance to alkylation damage. However, the global transcriptional and translational changes during this response have not been reported. Here we present time-dependent global gene and protein expression profiles following treatment with methyl methanesulfonate (MMS) in E. coli W3110 and its ada mutant strains.ResultsTranscriptome profiling showed that 1138 and 2177 genes were differentially expressed in response to MMS treatment in the wild-type and mutant strains, respectively. A total of 81 protein spots representing 76 nonredundant proteins differentially expressed were identified using 2-DE and LC-MS/MS. In the wild-type strain, many genes were differentially expressed upon long-exposure to MMS, due to both adaptive responses and stationary phase responses. In the ada mutant strain, the genes involved in DNA replication, recombination, modification and repair were up-regulated 0.5 h after MMS treatment, indicating its connection to the SOS and other DNA repair systems. Interestingly, expression of the genes involved in flagellar biosynthesis, chemotaxis, and two-component regulatory systems related to drug or antibiotic resistance, was found to be controlled by Ada.ConclusionThese results show in detail the regulatory components and pathways controlling adaptive response and how the related genes including the Ada regulon are expressed with this response.

Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1 Delta) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1 Delta strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1 Delta cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1 Delta mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Methylmethane Sulfonate Increases the Level of Superoxide Anions in Yeast Cells

ABSTRACTTy1 is a Saccharomyces cerevisiae retrotransposon with life cycle and structure very similar to the known oncoviruses. The transposition of the Ty1 mobile genetic element is induced by different stress conditions and chemical agents. Mutagens and carcinogens activate Ty1 elements for retrotransposition by certain types of genome injuries through DNA-damage signaling pathways but the mechanism of transcriptional activation by DNA damage has not been enough elucidated. Many of the activators of Ty1 mobility are powerful inducers of oxidative stress. Reactive oxygen species (ROS) can generate a variety of DNA lesions and can lead to a significant activation of Ty1 mobility. Recent data indicate that treatment of S. cerevisiae cells with the Ty1-mobility inducer methylmethane sulfonate (MMS) results in increased ROS levels. In response to MMS, both a direct DNA damaging effect and an enhanced ROS levels play role in the activation of Ty retrotransposition. The results obtained in this study provide evidence for a dependence of Ty1 retrotranspsoition on increased production of ROS. The increased ROS levels may have an independent and a key role in the induction of Ty1 retrotransposition by MMS. We suppose that MMS treatment of cells with functioning mitochondrial oxidative phosphorylation induces Ty1 retrotransposition by both, increased ROS levels and direct DNA damages, and the increased ROS levels seems to have the major role in the induction of Ty1 mobility.

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase–double Holliday junction dissolvasome

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.

Kinetics of γ‐H2AX focus formation upon treatment of cells with UV light and alkylating agents

Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

A Potential Link between Alternative Splicing of theNBS1Gene and DNA Damage/Environmental Stress

NBS1 forms a multimetric complex with MRE11/RAD50, which acts as the sensor of DNA double-strand breaks (DSBs). The mechanisms controlling the expression of NBS1 remain largely unknown. Here we show that NBS1 is transcribed as both a wild-type and an alternatively spliced form exhibiting a premature stop codon in an alternative 50-bp exon in intron 2. Although the wild-type transcript predominates in most tissues, the spliced transcript is abundant in resting peripheral blood mononuclear cells (PBMCs). Levels of the spliced form of NBS1 decreased rapidly after irradiation as levels of the wild-type NBS1 transcript increased, resulting in increased levels of NBS1 protein. Both mitogenic stimulation and methyl methanesulfonate treatment also altered the splicing pattern of NBS1. Resting PBMCs, which predominantly express spliced NBS1, were more susceptible to radiation than mitogen-stimulated cells, which showed predominant expression of the wild-type transcript. Since the alternatively spliced NBS1 gene likely did not produce protein, this alternative splicing seems to be associated with the control of NBS1 protein. Thus alternative splicing of the NBS1 gene may be associated with the regulation of NBS1 in response to DSBs, DNA alkylation damage, and mitogenic response.

Rpn4p is a positive and negative transcriptional regulator of the ubiquitin-proteasome system

Ubiquitin-proteasome proteolytic system participates in metabolism of the majority of intracellular proteins and regulation of key cellular processes in eukaryotes. While the structure and functioning of this system is studied rather well, a little is known about regulation of its genes expression. At present time, the only regulatory system of transcription of proteasome genes is found in the yeast Saccharomyces cerevisiae. This system includes Rpn4p-proteasome-associated transcriptional regulator and its binding site called PACE (Proteasome Associated Control Element). To learn more about function of Rpn4p as a transcriptional regulator, there are following questions: 1) is the Rpn4p regulator for PACE-containing genes which encode for components of protein ubiquitinylation system 2) what is the contribution of Rpn4p in stress-activated level of mRNA of proteasome genes. In this work, using semiquantitative RT-PCR we have shown that deletion of RPN4 gene leads to decreasing in mRNA level of the genes of ubiqitination system RAD6, RAD23 and CDC48, while UBI4 mRNA level is increased in this strain. In the presence of alkylating agent methyl methanesulfonate or under heat shock we observed Rpn4 p-dependent elevation of mRNA level of the proteasomal genes RPT4 and RPNS. At the same time, CDC48 mRNA level is decreased in wild type yeast strain upon methyl methanesulfonate treatment. These data indicate that under normal or stress conditions Rpn4p may act as an activator or repressor for the genes of the ubiquitin-proteasome system.