Polymerase Lambda Research Articles

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

Abstract PO3-26-09: A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities

Abstract Despite the development of screening techniques and therapies, nearly 30% of breast cancers progress to Stage IV metastatic disease. These advanced cancers are highly proliferative, engaging in ongoing DNA synthesis that requires ribonucleotide reductase (RNR), the rate-limiting enzyme for dNTP synthesis. RRM2, the small subunit of RNR, activates the enzyme, increases deoxynucleotide pools, and is overexpressed in many cancers, including breast cancer. Thus, RNR is an established therapeutic target in advanced breast cancer. Currently, gemcitabine is the only FDA-approved RNR inhibitor used to treat Stage IV disease. As a nucleoside analog, gemcitabine also causes severe dose-limiting toxicities (i.e. myelosuppression and hepatotoxicity) due to off-target DNA replication termination. We hypothesize that a novel, non-nucleoside, RNR inhibitor may overcome these limitations. To address this, we developed TMU27a, a derivative of naphthyl salicylic acyl hydrazone (NSAH), which was identified in an in silico screen for its high-affinity binding to the RNR catalytic site. To measure direct interactions between TMU27a and purified RNR protein, we used surface plasmon resonance, revealing on-target, tight binding (Kd = 150 nM). Unbiased docking and molecular dynamics simulations (using lowest energy calculations) predicted the binding sight of TMU27a to be the catalytic site of RNR, where it is stabilized through extensive interactions formed with conserved residues. Preliminary docking predictions also indicate multiple clash sites between TMU27a and the DNA polymerase lambda nucleotide pocket even in the lowest energy pose (unlike gemcitabine), suggesting that TMU27a inhibits RNR activity through a novel mechanism without significant off-target binding to other nucleotide binding proteins that contribute to toxicity. Demonstrating on-target inhibition, TMU27a reduces dATP levels and induces S and G2/M-phase stalling, leading to significant DNA damage and chromosomal instability. This excessive genomic damage causes growth repression, apoptosis, and senescence in several triple negative breast cancer (TNBC) cell lines. In xenograft mouse models of TNBC, TMU27a fully suppressed tumor growth without impacting mouse weight, suggesting strong drug efficacy in the absence of overt toxicity. Moreover, while short-term exposure to gemcitabine suppresses red and white blood cell populations, TMU27a does not. Together, these data indicate that TMU27a and its potential analogs are novel, non-toxic RNR inhibitors that overcome the limitations of gemcitabine for treating breast cancer. We are currently investigating how and where TMU27a binds human RNR using cryo-electron microscopy with the goal of generating a novel class of RNR inhibitors through structure-based drug design, improving binding affinity and selectivity. Citation Format: Taylor Baker, Kristen Weber-Bonk, Wei Huang, Derek Taylor, Ruth Keri. A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-09.

DNA polymerase λ Loop1 variant yields unexpected gain-of-function capabilities in nonhomologous end-joining

DNA polymerases lambda (Polλ) and mu (Polμ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polμ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end—albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polμ—synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.

Immune boosting and ameliorative properties of aqueous extract of Vernonia amygdalina Delile against MSG-induced genotoxicity: An in silico and in vivo approach

Vernonia amygdalina (VA) is popularly consumed as food and as medicine due to its nutritional and bioactive constituents. This study assessed the anti-genotoxic effect of aqueous leaf extract of VA against monosodium (MSG) –induced genotoxicity. Crude extraction and phytochemical analysis were done using standard methods. In silico studies was done using compounds in the extract against Bcl-2, NF-kB 50, DNA polymerase lambda, DNA ligase, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Twelve rats were divided into three groups with four rats in each group. Group I was fed on food and water, group II received MSG (4 g/kg) per body weight (pbw) intraperitoneally, group III received MSG (4 g/kg) pbw intraperitoneally followed by oral dose of VA leaf extract (250 mg/kg) per body weight. The number of the micronucleated red blood cells and white blood cells were determined from blood smears microscopically. Results showed that aqueous extract of VA contained in mg/100 g alkaloids (7.04 ± 0.16), saponins (3.91 ± 0.13), flavonoid (1.64 ± 0.16), phenol (3.40 ± 0.12) and tannins (0.07 ± 0.32). In silico studies revealed high binding interaction (ΔG > −8.6) of vernoniosides D and E with all the tested proteins. There was a reduction in the number of micronucleated cells, neutrophils and eosinophils of the treated group compared to the MSG group, while there was an increase in the lymphocyte count. The anti-genotoxic effects of VA leaf extract might be attributed to the synergistic interaction of the various bioactive components in the extract. VA could be a potential plant for the prevention of cancer and other diseases that attenuate the immune system.

Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination.

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

RanBP2-Mediated SUMOylation Promotes Human DNA Polymerase Lambda Nuclear Localization and DNA Repair

Cellular DNA is under constant attack by a wide variety of agents, both endogenous and exogenous. To counteract DNA damage, human cells have a large collection of DNA repair factors. Among them, DNA polymerase lambda (Polλ) stands out for its versatility, as it participates in different DNA repair and damage tolerance pathways in which gap-filling DNA synthesis is required. In this work, we show that human Polλ is conjugated with Small Ubiquitin-like MOdifier (SUMO) proteins both in vitro and in vivo, with Lys27 being the main target of this covalent modification. Polλ SUMOylation takes place in the nuclear pore complex and is mediated by the E3 ligase RanBP2. This post-translational modification promotes Polλ entry into the nucleus, which is required for its recruitment to DNA lesions and stimulated by DNA damage induction. Our work represents an advance in the knowledge of molecular pathways that regulate cellular localization of human Polλ, which are essential to be able to perform its functions during repair of nuclear DNA, and that might constitute an important point for the modulation of its activity in human cells.

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.

Variations in nuclear localization strategies among pol X family enzymes.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

DNA polymerase beta participates in DNA End-joining.

DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol β), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.

Living on the Edge: DNA Polymerase Lambda between Genome Stability and Mutagenesis.

In human cells, only four DNA polymerases (pols) are necessary and sufficient for the duplication of the genetic information. However, more than a dozen DNA pols are required to maintain its integrity. Such a high degree of specialization makes DNA repair pols able to cope with specific lesions or repair pathways. On the other hand, the same DNA pols can have partially overlapping roles, which could result in possible conflicts of functions, if the DNA pols are not properly regulated. DNA pol λ is a typical example of such an enzyme. It is a multifunctional enzyme, endowed with special structural and biochemical properties, which make it capable of participating in different DNA repair pathways such as base excision repair, nonhomologous end joining, and translesion synthesis. However, when mutated or deregulated, DNA pol λ can also be a source of genetic instability. Its multiple roles in DNA damage tolerance and its ability in promoting tumor progression make it also a possible target for novel anticancer approaches.

Abstract 2492: DNA polymerase beta participates in DNA end-joining

Abstract DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA polymerase theta (Pol θ), an A-family polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family polymerases, mammalian DNA polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA polymerase beta (Pol β), another X-family polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair. Citation Format: Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. DNA polymerase beta participates in DNA end-joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2017-2492

Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response

Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

Estrogen Drives Cellular Transformation and Mutagenesis in Cells Expressing the Breast Cancer-Associated R438W DNA Polymerase Lambda Protein.

The Pol λ R438W mutation can serve as a biomarker to predict cancer risk and implicates that treatment with estrogen in individuals with this mutation may further increase their risk of breast cancer. Mol Cancer Res; 14(11); 1068-77. ©2016 AACR.

A fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG.

8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.

Editorial: Maintenance of Genome Integrity: DNA Damage Sensing, Signaling, Repair, and Replication in Plants.

Editorial: Maintenance of Genome Integrity: DNA Damage Sensing, Signaling, Repair, and Replication in Plants.

Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda.

8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

In-silico prediction of drug targets, biological activities, signal pathways and regulating networks of dioscin based on bioinformatics.

BackgroundInverse docking technology has been a trend of drug discovery, and bioinformatics approaches have been used to predict target proteins, biological activities, signal pathways and molecular regulating networks affected by drugs for further pharmacodynamic and mechanism studies.MethodsIn the present paper, inverse docking technology was applied to screen potential targets from potential drug target database (PDTD). Then, the corresponding gene information of the obtained drug-targets was applied to predict the related biological activities, signal pathways and processes networks of the compound by using MetaCore platform. After that, some most relevant regulating networks were considered, which included the nodes and relevant pathways of dioscin.Results71 potential targets of dioscin from humans, 7 from rats and 8 from mice were screened, and the prediction results showed that the most likely targets of dioscin were cyclin A2, calmodulin, hemoglobin subunit beta, DNA topoisomerase I, DNA polymerase lambda, nitric oxide synthase and UDP-N-acetylhexosamine pyrophosphorylase, etc. Many diseases including experimental autoimmune encephalomyelitis of human, temporal lobe epilepsy of rat and ankylosing spondylitis of mouse, may be inhibited by dioscin through regulating immune response alternative complement pathway, G-protein signaling RhoB regulation pathway and immune response antiviral actions of interferons, etc. The most relevant networks (5 from human, 3 from rat and 5 from mouse) indicated that dioscin may be a TOP1 inhibitor, which can treat cancer though the cell cycle– transition and termination of DNA replication pathway. Dioscin can down regulate EGFR and EGF to inhibit cancer, and also has anti-inflammation activity by regulating JNK signaling pathway.ConclusionsThe predictions of the possible targets, biological activities, signal pathways and relevant regulating networks of dioscin provide valuable information to guide further investigation of dioscin on pharmacodynamics and molecular mechanisms, which also suggests a practical and effective method for studies on the mechanism of other chemicals.