Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

Improvement in organic solvent resistance of keratinase BLk by directed evolution.

Keratinase, a vital enzyme in hair degradation, requires enhanced stability for industrial applications in the harsh reaction environment used for keratin hydrolysis. Previous studies have focused on improving keratinase thermostability. In this study, directed evolution was applied to enhance the organic solvent stability of the keratinase BLk from Bacillus licheniformis. Three mutants were identified, exhibiting significant enhanced stability in various solvents, although no similar improvements were observed in terms of thermostability. The identified mutations were located on the enzyme surface. The half-lives of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to that of the wild type (WT). Similarly, in the presence of 50% (v/v) acetone, the half-lives of these mutants increased by 22-, 27-, and 27-fold compared to that of the WT enzyme. Notably, the proteolytic activity of all the selected mutants was similar to that of the WT enzyme. Furthermore, molecular dynamics simulation was used to assess the possible reasons for enhanced solvent stability. These results suggest that heightened intramolecular interactions, such as hydrogen bonding and hydrophobic interactions, contribute to improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.

Phosphorylation Regulation of a Histone-like HU Protein from Deinococcus radiodurans.

BACKGROUND: Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU. OBJECTIVE: We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans. METHODS: LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses. RESULTS AND DISCUSSION: The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress. CONCLUSIONS: Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.

Pol µ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol µ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol µ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol µ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol µ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.

MazEF Toxin-Antitoxin System-Mediated DNA Damage Stress Response in Deinococcus radiodurans.

Deinococcus radiodurans shows marked resistance to various types of DNA-damaging agents, including mitomycin C (MMC). A type II toxin-antitoxin (TA) system that responds to DNA damage stress was identified in D. radiodurans, comprising the toxin MazF-dr and the antitoxin MazE-dr. The cleavage specificity of MazF-dr, an endoribonuclease, was previously characterized. Here, we further investigated the regulatory role of the MazEF system in the response to DNA damage stress in D. radiodurans. The crystal structure of D. radiodurans MazF (MazF-dr) was determined at a resolution of 1.3 Å and is the first structure of the toxin of the TA system of D. radiodurans. MazF-dr forms a dimer mediated by the presence of interlocked loops. Transcriptional analysis revealed 650 downregulated genes in the wild-type (WT) strain, but not in the mazEF mutant strain, which are potentially regulated by MazEF-dr in response to MMC treatment. Some of these genes are involved in membrane trafficking and metal ion transportation. Subsequently, compared with the WT strain, the mazEF mutant strain exhibited much lower MMC-induced intracellular iron concentrations, reactive oxygen species (ROS), and protein carbonylation levels. These results provide evidence that MazEF-mediated cell death in D. radiodurans might be caused by an increase in ROS accumulation upon DNA damage stress.

Late embryogenesis abundant group3 protein (DrLEA3) is involved in antioxidation in the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is able to survive under extreme conditions, including high doses of ionizing radiation, desiccation and oxidative stress. In addition to enhanced DNA repair capabilities, an effective antioxidation system plays an important role in its robustness. Previous studies have linked the radiation resistance of D. radiodurans to its prolonged desiccation tolerance phenotype, which both cause DNA damage. In the current study, we investigated the roles of dr_1172 in D. radiodurans, the gene encoding a typical group 3 LEA protein (DrLEA3) conserved within Deinococcus species. In addition to the increased transcriptional level under oxidative stress, the inactivation of dr_1172-sensitized cells to H2O2 treatments and the reduced cellular antioxidation activities suggested that dr_1172 is involved in the cellular defense against oxidative stress. Moreover, DrLEA3 was enriched at the cell membrane and bound to various types of metal ions. Cells devoid of DrLEA3 showed a decreased intracellular Mn/Fe concentration ratio, indicating that DrLEA3 also plays a role in maintaining metal ion homeostasis in vivo.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in the Extreme Resistance of Deinococcus radiodurans.

Increasing evidence shows that the succinylation of lysine residues mainly regulates enzymes involved in the carbon metabolism pathway, in both prokaryotic and eukaryotic cells. Deinococcus radiodurans is one of the most radioresistant organisms on earth and is famous for its robust resistance. A major goal in the current study of protein succinylation is to explore its function in D. radiodurans. High-resolution LC-MS/MS is used for qualitative proteomics to perform a global succinylation analysis of D. radiodurans and 492 succinylation sites in 270 proteins are identified. These proteins are involved in a variety of biological processes and pathways. It is found that the enzymes involved in nucleic acid binding/processing are enriched in D. radiodurans compared with their previously reported levels in other bacteria. The mutagenesis studies confirm that succinylation regulates the enzymatic activities of species-specific proteins PprI and DdrB, which belong to the radiation-desiccation response regulon. Together, these results provide insight into the role of lysine succinylation in the extreme resistance of D. radiodurans.