The Nuclease Activity of the Yeast Dna2 Protein, Which Is Related to the RecB-like Nucleases, Is Essential in Vivo

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype

Biochemical analysis of human Dna2

Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5'–3' nuclease activity with preference for single-stranded 5' flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5' end. Moreover, hDna2 shows strong 3'–5' nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5'–3' activity, is suppressed by steric hindrance at the 3'-end, suggesting that the 3'–5' nuclease requires a 3' single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3'–5' nuclease activity may be more important than previously thought

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway

Fluormetric assay for nuclease activity

Cytochemical studies of planetary microorganisms - deoxyribonuclease /dnase/ assay, fluorescein reactions, and enzyme activit

Genetic separation of Sae2 nuclease activity from Mre11 nuclease functions in budding yeast

Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities

Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc-finger proteins

AbstractThe CCCH class of zinc fingers occurs in a large number of Arabidopsis proteins. Previous studies revealed that one such protein is a nuclease, the activity of which is attributable to one of the CCCH motifs. To examine whether nuclease activity is a more general characteristic of CCCH zinc finger containing proteins, five other such Arabidopsis proteins were assayed for a similar activity. The results indicate that all of these proteins possess nuclease activity. Thus, nuclease activity may be a common characteristic of Arabidopsis CCCH-containing proteins

Improving Fab’ fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA

OBJECTIVES: To reduce unwanted Fab’ leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab’ fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. RESULTS: We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab’ grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab’ coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab’ leakage relative to the original autonucleolytic Fab’ strain with OmpA-fused staphylococcal nuclease. CONCLUSIONS: We successfully rescued Fab’ leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab’ fragment to the surrounding growth media

Structural basis for the nuclease activity of a bacteriophage large terminase

The DNA-packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X-ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structure-based alignment with the distantly related T4 gp17 terminase shows the conservation of an extended β-sheet and an auxiliary β-hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the β-hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the β-hairpin, altering active site access and the orientation of catalytically essential residues

The MRX Complex Plays Multiple Functions in Resection of Yku- and Rif2-Protected DNA Ends

The ends of both double-strand breaks (DSBs) and telomeres undergo tightly regulated 5′ to 3′ resection. Resection of DNA ends, which is specifically inhibited during the G1 cell cycle phase, requires the MRX complex, Sae2, Sgs1 and Exo1. Moreover, it is negatively regulated by the non-homologous end-joining component Yku and the telomeric protein Rif2. Here, we investigate the nuclease activities that are inhibited at DNA ends by Rif2 and Yku in G1 versus G2 by using an inducible short telomere assay. We show that, in the absence of the protective function of Rif2, resection in G1 depends primarily on MRX nuclease activity and Sae2, whereas Exo1 and Sgs1 bypass the requirement of MRX nuclease activity only if Yku is absent. In contrast, Yku-mediated inhibition is relieved in G2, where resection depends on Mre11 nuclease activity, Exo1 and, to a minor extent, Sgs1. Furthermore, Exo1 compensates for a defective MRX nuclease activity more efficiently in the absence than in the presence of Rif2, suggesting that Rif2 inhibits not only MRX but also Exo1. Notably, the presence of MRX, but not its nuclease activity, is required and sufficient to override Yku-mediated inhibition of Exo1 in G2, whereas it is required but not sufficient in G1. Finally, the integrity of MRX is also necessary to promote Exo1- and Sgs1-dependent resection, possibly by facilitating Exo1 and Sgs1 recruitment to DNA ends. Thus, resection of DNA ends that are protected by Yku and Rif2 involves multiple functions of the MRX complex that do not necessarily require its nuclease activity

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions