SUMOylation of yeast Pso2 enhances its translocation and accumulation in the mitochondria and suppresses methyl methanesulfonate-induced mitochondrial DNA damage.

Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria. Reciprocally, SUMOylation had no discernible impact on Pso2 translocation to the nucleus, despite the presence of steady-state levels of SUMOylated Pso2 across the cell cycle. Furthermore, substitution of the invariant residues K97 and K575 by arginine in the Pso2 SUMO consensus motifs severely impaired SUMOylation and abolished its translocation to the mitochondria of MMS-treated wild type cells, but not in unstressed cells. We demonstrate that whilst Siz1 and Siz2 SUMO E3 ligases catalyze Pso2 SUMOylation, the former plays a dominant role. Notably, we found that the phenotypic characteristics of the SUMOylation-defective mutant Pso2K97R/K575R closely mirrored those observed in the Pso2Δ petite mutant. Additionally, leveraging next-generation sequencing analysis, we demonstrate that Pso2 mitigates MMS-induced damage to mitochondrial DNA (mtDNA). Viewed together, our work offers previously unknown insights into the link between genotoxic stress-induced SUMOylation of Pso2 and its preferential targeting to the mitochondria, as well as its role in attenuating MMS-induced mtDNA damage.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Xrs2 Dependent and Independent Functions of the Mre11-Rad50 Complex.

The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, we found that end resection in its absence remains Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1/ATM kinase signaling and non-homologous end joining, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR complex and other repair proteins.

XRCC1 counteracts poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib, and a clinical alkylating agent, temozolomide, by promoting the removal of trapped PARP1 from broken DNA.

Base excision repair (BER) removes damaged bases by generating single-strand breaks (SSBs), gap-filling by DNA polymerase ß (POLß), and resealing SSBs. A base-damaging agent, methyl methanesulfonate (MMS) is widely used to study BER. BER increases cellular tolerance to MMS, anti-cancer base-damaging drugs, temozolomide, carmustine, and lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLß to SSBs, but XRCC1-/- cells are much more sensitive to MMS than PARP1-/- cells. We recently report that the PARP1 loss in XRCC1-/- cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1-/- cells also restores their tolerance to the three anti-cancer base-damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1-/- mutation, but not POLß-/- , with olaparib and talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis-dependent and independent mechanisms.

Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast.

Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.

Expression and Characterization of the Staphylococcus aureus RecA protein: A mapping of canonical functions.

Recombinases are responsible for homologous recombination (HR), proper genome maintenance, and accurate deoxyribonucleic acid (DNA) duplication. Moreover, HR plays a determining role in DNA transaction processes such as DNA replication, repair, recombination, and transcription. Staphylococcus aureus, an opportunistic pathogen, usually causes respiratory infections such as sinusitis, skin infections, and food poisoning. To date, the role of the RecA gene product in S. aureus remains obscure. In this study, we attempted to map the functional properties of the RecA protein. S. aureus expresses the recA gene product in vivo upon exposure to the DNA-damaging agents, ultraviolet radiation, and methyl methanesulfonate. The recombinant purified S. aureus RecA protein displayed strong single-stranded DNA affinity compared to feeble binding to double-stranded DNA. Interestingly, the RecA protein is capable of invasion and formed displacement loops and readily performed strand-exchange activities with an oligonucleotide-based substrate. Notably, the S. aureus RecA protein hydrolyzed the DNA-dependent adenosine triphosphate and cleaved LexA, showing the conserved function of coprotease. This study provides the functional characterization of the S. aureus RecA protein and sheds light on the canonical processes of homologous recombination, which are conserved in the gram-positive foodborne pathogen S. aureus.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate.

The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.

Influence of Kv11.1 (hERG1) K+ channel expression on DNA damage induced by the genotoxic agent methyl methanesulfonate.

Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K+ channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.

Homolog of Saccharomyces cerevisiae SLX4 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

SLX4 is a scaffold to coordinate the action of structure-specific endonucleases that are required for homologous recombination and DNA repair. In view of ScSLX4 functions in the maintenance and stability of the genome in Saccharomyces cerevisiae, we have explored the roles of CaSLX4 in Candida albicans. Here, we constructed slx4Δ/Δ mutant and found that it exhibited increased sensitivity to the DNA damaging agent, methyl methanesulfonate (MMS) but not the DNA replication inhibitor, hydroxyurea (HU). Accordingly, RT-qPCR and western blotting analysis revealed the activation of SLX4 expression in response to MMS. The deletion of SLX4 resulted in a defect in the recovery from MMS-induced filamentation to yeast form and re-entry into the cell cycle. Like many other DNA repair genes, SLX4 expression was activated by the checkpoint kinase Rad53 under MMS-induced DNA damage. In addition, SLX4 was not required for the inactivation of the DNA damage checkpoint, as indicated by normal phosphorylation of Rad53 in slx4Δ/Δ cells. Therefore, our results demonstrate SLX4 plays an important role in cell recovery from MMS-induced DNA damage in C. albicans.

Yeast-based screening of cancer mutations in the DNA damage response protein Mre11 demonstrates importance of conserved capping domain residues.

DNA damage response (DDR) pathways are initiated to prevent mutations from being passed on in the event of DNA damage. Mutations in DDR proteins can contribute to the development and maintenance of cancer cells, but many mutations observed in human tumors have not been functionally characterized. Because a proper response to DNA damage is fundamental to living organisms, DDR proteins and processes are often highly conserved. The goal of this project was to use Saccharomyces cerevisiae as a model for functional screening of human cancer mutations in conserved DDR proteins. After comparing the cancer mutation frequency and conservation of DDR proteins, Mre11 was selected for functional screening. A subset of mutations in conserved residues was analyzed by structural modeling and screened for functional effects in yeast Mre11. Yeast expressing wild type or mutant Mre11 were then assessed for DNA damage sensitivity using hydroxyurea (HU) and methyl methanesulfonate (MMS). The results were further validated in human cancer cells. The N-terminal point mutations tested in yeast Mre11 do not confer sensitivity to DNA damage sensitivity, suggesting that these residues are dispensable for yeast Mre11 function and may have conserved sequence without conserved function. However, a mutation near the capping domain associated with breast and colorectal cancers compromises Mre11 function in both yeast and human cells. These results provide novel insight into the function of this conserved capping domain residue and demonstrate a framework for yeast-based screening of cancer mutations.

Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses.

Poly(ADP-ribos)ylation (PARylation) is a reversible posttranslational modification found in higher eukaryotes. However, little is known about PARylation acceptor proteins. Here, we describe a sensitive proteomics approach based on high-accuracy quantitative mass spectrometry for the identification of PARylated proteins induced under different cellular stress conditions. While confirming the majority of known PARylated substrates, our screen identifies numerous additional PARylation targets. In vivo and in vitro validation of acceptor proteins confirms that our methodology targets covalent PARylation. Nuclear proteins encompassing nucleic acid binding properties are prominently PARylated upon genotoxic stress, consistent with the nuclear localization of ARTD1/PARP1 and ARTD2/PARP2. Distinct differences in proteins becoming PARylated upon various genotoxic insults are observed, exemplified by the PARylation of RNA-processing factors THRAP3 and TAF15 under oxidative stress. High-content imaging reveals that PARylation affects the nuclear relocalization of THRAP3 and TAF15, demonstrating the potential of our approach to uncover hitherto unappreciated processes being controlled by specific genotoxic-stress-induced PARylation.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and in vivo.

Interest in pharmacological agents capable of increasing cellular NAD+ concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD+ concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD+ concentrations by 2.5-10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD+ increases at equivalent concentrations. NRH also provides substantial NAD+ increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD+/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD+-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD+ consumption, suggesting that it acts as a biochemical precursor to NAD+ Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD+ and a potent pharmacologic agent for NAD+ concentration enhancement in cells and tissues.

SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair.

XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER.

In Situ Alkylation of Reconstructed Human Epidermis by Methyl Methanesulfonate: A Quantitative HRMAS NMR Chemical Reactivity Mapping.

Allergic contact dermatitis (ACD) is a reaction of the immune system resulting from skin sensitization to an exogenous hazardous chemical and leading to the activation of antigen-specific T-lymphocytes. The adverse outcome pathway (AOP) for skin sensitization identified four key events (KEs) associated with the mechanisms of this pathology, the first one being the ability of skin chemical sensitizers to modify epidermal proteins to form antigenic structures that will further trigger the immune system. So far, these interactions have been studied in solution using model nucleophiles such as amino acids or peptides. As a part of our efforts to better understand chemistry taking place during the sensitization process, we have developed a method based on the use of high-resolution magic angle spinning (HRMAS) NMR to monitor in situ the reactions of 13C substituted chemical sensitizers with nucleophilic amino acids of epidermal proteins in reconstructed human epidermis. A quantitative approach, developed so far for liquid NMR applications, has not been developed to our knowledge in a context of a semisolid nonanisotropic environment like the epidermis. We now report a quantitative chemical reactivity mapping of methyl methanesulfonate (MMS), a sensitizing methylating agent, in reconstructed human epidermis by quantitative HRMAS (qHRMAS) NMR. First, the haptenation process appeared to be much faster in RHE than in solution with a maximum concentration of adducts reached between 4 and 8 h. Second, it was observed that the concentration of cysteine adducts did not significantly increase with the dose (2.07 nmol/mg at 0.4 M and 2.14 nmol/mg at 1 M) nor with the incubation time (maximum of 2.27 nmol/mg at 4 h) compared to other nucleophiles, indicating a fast reaction and a potential saturation of targets. Third, when increasing the exposure dose, we observed an increase of adducts up to 12.5 nmol/mg of RHE, excluding cysteine adducts, for 3112 µg/cm2 (1 M solution) of (13C)MMS. This methodology applied to other skin sensitizers could allow for better understanding of the potential links between the amount of chemical modifications formed in the epidermis in relation to exposure and the sensitization potency.

Proteomics Study of DNA-Protein Crosslinks in Methylmethanesulfonate and Fe2+-EDTA-Exposed Human Cells.

The formation of covalently bound DNA-protein crosslinks (DPCs) is linked to the pathophysiology of cancers and many other degenerative diseases. Knowledge of the proteins that were frequently involved in forming DPCs will improve our understanding of the etiological mechanism of diseases and facilitate the establishment of preventive measures and treatment methods. By using SDS-PAGE and nano-LC coupled Orbitrap LC-MS/MS analyses, we identified, for the first time, that the major DNA-cross-linked proteins in HeLa cells exposed to a methylating agent (methylmethanesulfonate) or hydroxyl free radicals are transcription-associated proteins. In particular, histone H2B3B and poly(rC) binding protein 2 were identified as the most frequent DPC-forming proteins.

Mitochondrial behavior during nuclear and mitochondrial DNA repair in Trypanosoma cruzi epimastigotes.

Different genotoxic agents can lead to DNA single- and double-strand breaks, base modification and oxidation. As most living organisms, Trypanosoma cruzi is subjected to oxidative stress during its life cycle; thus, DNA repair is essential for parasite survival and establishment of infection. The mitochondrion plays important roles beyond the production of ATP. For example, it is a source of signaling molecules, such as the superoxide anion and H2O2. Since T. cruzi has only one mitochondrion, the integrity of this organelle is pivotal for parasite viability. H2O2 and methyl methanesulfonate cause DNA lesions in T. cruzi that are repaired by different DNA repair pathways. Herein, we evaluate mitochondrial involvement during the repair of nuclear and mitochondrial DNA in T. cruzi epimastigotes incubated with these two genotoxic agents under conditions that induce repairable DNA damage. Overall, in both treatments, an increase in oxygen consumption rates and in mitochondrial H2O2 release was observed, as well as maintenance of ATP levels compared to control. Interestingly, these changes coincided with DNA repair kinetics, suggesting the importance of the mitochondrion for this energy-consuming process.

Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1 locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

Evaluation of the Effect of p-Tyrosol on the Level of DNA Damage in the DNA Comet Assay In Vivo.

The effect of p-tyrosol on the spontaneous level of DNA damage in the cells of the bone marrow, liver, kidney, and rectum of mice (series I) and on the genotoxic effects of cytostatic drugs with different mechanisms of action in rat testicular cells (series II) was studied by DNA comet assay on C57BL/6 mice. p-Tyrosol was administered in a dose of 40 mg/kg once (series I) or for 5 days before and 5 days after cytostatic exposure (busulfan, paclitaxel, methotrexate; series II). It was found that p-tyrosol reduced spontaneous level of DNA damage in all studied organs. p-Tyrosol exhibited an antigenotoxic effect with respect to the DNA-damaging action of methotrexate and produced no genoprotective effect in case of busulfan and paclitaxel.