Excessive use of pesticides in agriculture has led to several problems pertaining to loss of soil fertility and environmental degradation. Biological control agents offer the best alternative to ...reduce use of toxic pesticides.
Paenibacillus sp. D1 isolated from the effluent treatment plant of a seafood processing industry exhibited broad spectrum tolerance towards a number of pesticides at concentrations higher than recommended for field applications. The isolate showed enhanced growth and chitinase production in the presence of some protectant fungicides. None of the tested demethylase inhibitor (DMI) fungicides inhibited growth and chitinase production except triadimefon. The isolate was also tolerant to most commonly used insecticides belonging to the organophosphate, carbamate and cyclodiene organochloride classes. Chitinase of
Paenibacillus sp. D1 was found to be more tolerant than the organism itself and was highly stable in the presence of pesticides at the temperature under field conditions in Gujarat, India, i.e. 40
°C. This was suggestive of its potential in integrated pest management (IPM) to significantly reduce the use of harmful chemicals. To our knowledge this is the first extensive study on pesticide tolerance of the
Paenibacillus species and its chitinase.
p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1’s function is well established in DNA double-strand break repair, how its role in p53 signaling ...is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.
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•AHNAK is a G1-enriched interactor of 53BP1•AHNAK controls 53BP1-mediated G1-S phase transition upon DNA damage•AHNAK restrains 53BP1 oligomerization and phase separation•AHNAK balances between apoptosis and senescence in cancer and non-transformed cells
Ghodke et al. identify the large scaffolding protein AHNAK as a G1-enriched interactor of 53BP1 that ensures optimal partitioning of 53BP1 into phase-separated condensates and limits excessive interaction with p53, which would otherwise lead to apoptosis in cancer cells and senescence in non-transformed cells.
Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair ...via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single- over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3′ single-stranded DNA overhang but not of 5′ overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.
Background: The mechanism of DSB end resection in yeast nuclease deficient mre11 mutants and Mre11 nuclease-independent ATM activation in mammalian cells remains unclear.
Results: Mre11 binds to DSB ends and also promotes end bridging. Rad50, Xrs2, and Sae2 potentiate Mre11-catalyzed DNA unwinding activity.
Conclusion: Mre11 nuclease activity is dispensable for DNA binding and unwinding activity.
Significance: These studies reveal a novel mechanism of processing of DSBs by MRX-Sae2 complex.
Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps ...remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end–joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination.
The HORMA domain (for Hop1p, Rev7p and MAD2) was discovered in three chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae. This domain has also been found in proteins with ...similar functions in organisms including plants, animals and nematodes. The HORMA domain containing proteins are thought to function as adaptors for meiotic checkpoint protein signaling and in the regulation of meiotic recombination. Surprisingly, new work has disclosed completely unanticipated and diverse functions for the HORMA domain containing proteins. A. M. Villeneuve and colleagues (Schvarzstein et al., 2013) show that meiosis-specific HORMA domain containing proteins plays a vital role in preventing centriole disengagement during Caenorhabditis elegans spermatocyte meiosis. Another recent study reveals that S. cerevisiae Atg13 HORMA domain acts as a phosphorylation-dependent conformational switch in the cellular autophagic process.
•HORMA domain is a protein domain found originally in chromatin-associated proteins.•HORMA containing proteins show a similar domain organization from yeast to humans.•HORMA domain plays a vital role in preventing centriole disengagement.•HORMA domain acts as a phosphorylation-dependent conformational switch in autophagy.•Here, we discuss the current knowledge about HORMA domain containing proteins.
In eukaryotes, the repair of DSBs is accomplished through two broadly defined processes: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The central step of HR is pairing and ...exchange of strands between two homologous DNA molecules, which is catalyzed by the conserved Rad51/RecA family of proteins. Prior to this step, an essential step in all HR pathways i.e. 5'→3' resection of broken DNA ends to generate 3' single stranded DNA tails. At the molecular level, initiation of DNA end resection is accomplished through the concerted action of MRX complex (Mre11, Rad50 and Xrs2) and Sae2 protein.
To elucidate the molecular basis underlying DSB end resection in S. cerevisiae mre11 nuclease deficient mutants, we have performed a comprehensive analysis of the role of S. cerevisiae Mre11 (henceforth called as ScMre11) in the processing of DSB ends using a variety of DNA substrates. We observed that S. cerevisiae Mre11(ScMre11) exhibits higher binding affinity for single- over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Furthermore, reconstitution of DSB end resection network in-vitro revealed that Rad50, Xrs2, and Sae2 potentiated the DNA unwinding activity of Mre11. Since the exonuclease activity of Mre11 is of the opposite polarity to that expected for resection of DSBs, unwinding activity of Mre11 in conjunction with Rad50, Xrs2, and Sae2 might provide an alternate mechanism for the generation of ssDNA intermediates for DSB end repair and HR. Additionally, ScMre11 displays strong homotypic as well as heterotypic interaction with Sae2. In summary, our results revealed important insights into the mechanism of DSB end processing and support a model in which Sae2, Rad50, and Xrs2 positively regulate the ScMre11-mediated DNA unwinding activity via their direct interactions or through allosteric effects on the DNA or cofactors.
Prompted by the closer association of MRX and Sae2 during DSB end processing, we asked whether Sae2 and its endonuclease activity is required for cellular response to replication stress caused by DNA damage. Toward this end, we examined the sensitivity of S. cerevisiae wild type, sae2Δ and various SAE2 mutant strains defective in phosphorylation and nuclease activity in the presence of different genotoxic agents, which directly or indirectly generate DSBs during replication. We found that S. cerevisiae lacking SAE2 show decreased cell viability, altered cell cycle dynamics after DNA damage, and more specifically, that Sae2 endonuclease activity is essential for these biological functions. To corroborate the genetic evidences for role of SAE2 during replicative stress, we investigated SAE2 functions in-vitro. For this, we purified native Sae2 protein and nuclease dead mutant of Sae2 i.e. sae2G270D. Our studies revealed dimeric forms of both the wild type and mutant forms of Sae2. Furthermore, Sae2 displays higher binding affinity and catalytic activity with branched DNA structures, such as Holliday junction and replication forks. By using nuclease dead Sae2 protein i.e. sae2G270D, we confirmed that the endonuclease activity is not fortuitous and is intrinsic to Sae2 polypeptide. Furthermore, nuclease-defective Mre11 stimulates Sae2endonuclease activity. Mapping of the cleavage sites of Sae2 revealed a distinct preference for cleavage on the 5' end of the Holliday junction, suggesting the importance of Sae2 nuclease during recombination mediated restart of the reversed replication fork. In summary, our data clearly demonstrate a previously uncharacterized role for Sae2 nuclease activity in resection of DSB ends, processing of intermediates of DNA replication/repair and attenuation of DNA replication stress-related defects in S. cerevisiae.
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