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The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by 60 Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling.

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg 2+ for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

Yeast Rad1–Rad10 (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4 , which encode proteins that interact physically with Rad1–Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1–Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1–Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1–Rad10 removes ICL via NER, whereas in S/G2, Rad1–Rad10 facilitates NER-independent replication-coupled ICLR. The yeast Rad1–Rad10 complex has multiple roles in DNA damage repair. Here the authors uncover mutants that uncouple the roles in UV excision repair and non-NER functions.

Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5′-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum. Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa2) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa2 was also studied, and 95.1% of GMPNa2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5–8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3′–5′ mode to produce 5′-oligonucleotides and 5′-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H 3 -thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

The structure-specific endonucleases Mus81-Mms4 and Yen1 are involved in the resolution of Holliday junctions and in crossover formation in the budding yeast. Now genetic work implicates the nucleotide excision repair nuclease complex Rad1-Rad10 in the processing of recombination intermediates formed between substrates with limited homology. Holliday junctions can be formed during homology-dependent repair of DNA double-strand breaks, and their resolution is essential for chromosome segregation and generation of crossover products. The Mus81-Mms4 and Yen1 nucleases are required for mitotic crossovers between chromosome homologs in Saccharomyces cerevisiae ; however, crossovers between dispersed repeats are still detected in their absence. Here we show that the Rad1-Rad10 nuclease promotes formation of crossover and noncrossover recombinants between ectopic sequences. Crossover products were not recovered from the mus81Δ rad1Δ yen1Δ triple mutant, indicating that all three nucleases participate in processing recombination intermediates that form between dispersed repeats. We suggest a new mechanism for crossovers that involves Rad1-Rad10 clipping and resolution of a single Holliday junction–containing intermediate by Mus81-Mms4 or Yen1 cleavage or by replication. Consistent with the model, we show accumulation of Rad1-dependent joint molecules in the mus81Δ yen1Δ mutant.

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2 % higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

Genome instability is a hallmark of cancer that can be caused by DNA replication stress. Copy number variation (CNV) is a type of genomic instability that has been associated with both tumorigenesis and drug resistance, but how these structural variants form in response to replication stress is not fully understood. Here, we established a direct repeat genetic reporter in Saccharomyces cerevisiae to detect recombination events that result in either a duplication or a deletion. Using this system, we measured recombination resulting from site-specific replication fork stalling initiated by Tus binding to an array of Ter sites. We found that a Tus/ Ter fork block downstream of direct repeats induced CNV by a mechanism involving the Mph1 translocase, Exo1-catalyzed end resection and Rad51-dependent strand invasion. While the Slx4 scaffold protein and its nuclease-binding partner, Rad1-Rad10, were shown to be required for duplications, we found that they suppress deletion formation in this context. These opposing functions suggest that both recombination products arise through a large loop heteroduplex intermediate that is cleaved by Rad1-Rad10 in a manner that promotes duplications and eliminates deletions. Taken together, these studies give insight into the mechanisms governing CNV in the context of replication fork stalling, which may ultimately provide a better understanding of how replication stress contributes to cancer and other diseases characterized by genome instability.

Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.