Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

Thymine DNA glycosylase regulates cell-cycle-driven p53 transcriptional control in pluripotent cells.

Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.

Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.

The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

OGG1: An emerging multifunctional therapeutic target for the treatment of diseases caused by oxidative DNA damage.

Oxidative DNA damage-related diseases, such as incurable inflammation, malignant tumors, and age-related disorders, present significant challenges in modern medicine due to their complex molecular mechanisms and limitations in identifying effective treatment targets. Recently, 8-oxoguanine DNA glycosylase 1 (OGG1) has emerged as a promising multifunctional therapeutic target for the treatment of these challenging diseases. In this review, we systematically summarize the multiple functions and mechanisms of OGG1, including pro-inflammatory, tumorigenic, and aging regulatory mechanisms. We also highlight the potential of OGG1 inhibitors and activators as potent therapeutic agents for the aforementioned life-limiting diseases. We conclude that OGG1 serves as a multifunctional hub; the inhibition of OGG1 may provide a novel approach for preventing and treating inflammation and cancer, and the activation of OGG1 could be a strategy for preventing age-related disorders. Furthermore, we provide an extensive overview of successful applications of OGG1 regulation in treating inflammatory, cancerous, and aging-related diseases. Finally, we discuss the current challenges and future directions of OGG1 as an emerging multifunctional therapeutic marker for the aforementioned challenging diseases. The aim of this review is to provide a robust reference for scientific researchers and clinical drug developers in the development of novel clinical targeted drugs for life-limiting diseases, especially for incurable inflammation, malignant tumors, and age-related disorders.

Thymine DNA glycosylase is an RNA-binding protein with high selectivity for G-rich sequences.

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.

Thymine DNA glycosylase mediates chromatin phase separation in a DNA methylation-dependent manner.

Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions in vitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model. We also provide evidence that TDG has the capacity to form phase-separated condensates in the cell nucleus. The ability of TDG to induce chromatin phase separation is dependent on its intrinsically disordered N- and C-terminal domains, which in isolation, promote the formation of chromatin-containing droplets having distinct physical properties, consistent with their unique mechanistic roles in the phase separation process. Interestingly, DNA methylation alters the phase behavior of the disordered domains of TDG and compromises formation of chromatin condensates by full-length TDG, indicating that DNA methylation regulates the assembly and coalescence of TDG-mediated condensates. Overall, our results shed new light on the formation and physical nature of TDG-mediated chromatin condensates, which have broad implications for the mechanism and regulation of TDG and its associated genomic processes.

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

The BCL11A transcription factor stimulates the enzymatic activities of the OGG1 DNA glycosylase.

The BCL11A transcription factor has previously been shown to interact with and stimulate the enzymatic activities of the NTHL1 DNA glycosylase and Pol ß polymerase. Here we show that BCL11A and a smaller peptide encompassing amino acids 160 to 520 can interact with the 8-oxoguanine DNA glycosylase, OGG1, increase the binding of OGG1 to DNA that contains an 8-oxoguanine base and stimulate the glycosylase activity of OGG1. Following BCL11A knockdown, we observed an increase in oxidized purines in the genome using comet assays, while immunoassays reveal an increase in 8-oxoG bases. Structure-function analysis indicates that the stimulation of OGG1 by BCL11A requires the zinc fingers 1, 2 and 3 as well as the proline-rich region between the first and second zing finger, but a glutamate-rich region downstream of zinc finger 3 is dispensable. Ectopic expression of a small peptide that contains the three zinc fingers can rescue the increase in 8-oxoguanine caused by BCL11A knockdown. These findings, together with previous results showing that BCL11A stimulates the enzymatic activities of NTHL1 and the Pol ß polymerase, suggest that high expression of BCL11A is important to protect cancer cells against oxidative DNA damage.

Uracil base PCR implemented for reliable DNA walking.

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

Repurposing sodium stibogluconate as an uracil DNA glycosylase inhibitor against prostate cancer using a time-resolved oligonucleotide-based drug screening platform.

Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

The Influence of Oxidized Imino-Allantoin in the Presence of OXOG on Double Helix Charge Transfer: A Theoretical Approach.

The genome is continuously exposed to a variety of harmful factors that result in a significant amount of DNA damage. This article examines the influence of a multi-damage site containing oxidized imino-allantoin (OXIa) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on the spatial geometry, electronic properties, and ds-DNA charge transfer. The ground stage of a d[A1OXIa2A3OXOG4A5]*d[T5C4T3C2T1] structure was obtained at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the condensed phase, with the energies obtained at the M06-2X/6-31++G** level. The non-equilibrated and equilibrated solvent-solute interactions were also considered. Theoretical studies reveal that the radical cation prefers to settle on the OXOG moiety, irrespective of the presence of OXIa in a ds-oligo. The lowest vertical and adiabatic ionization potential values were found for the OXOG:::C base pair (5.94 and 5.52 [eV], respectively). Conversely, the highest vertical and adiabatic electron affinity was assigned for OXIaC as follows: 3.15 and 3.49 [eV]. The charge transfers were analyzed according to Marcus' theory. The highest value of charge transfer rate constant for hole and excess electron migration was found for the process towards the OXOGC moiety. Surprisingly, the values obtained for the driving force and activation energy of electro-transfer towards OXIa2C4 located this process in the Marcus inverted region, which is thermodynamically unfavorable. Therefore, the presence of OXIa can slow down the recognition and removal processes of other DNA lesions. However, with regard to anticancer therapy (radio/chemo), the presence of OXIa in the structure of clustered DNA damage can result in improved cancer treatment outcomes.

DNA Mutagenicity of Hydroxyhydroquinone in Roasted Coffee Products and Its Suppression by Chlorogenic Acid, a Coffee Polyphenol, in Oxidative-Damage-Sensitive SAMP8 Mice.

Hydroxyhydroquinone (HHQ) is an oxidative component produced by roasting coffee beans and has been reported to generate relatively large amounts of reactive oxygen species (ROS). In this study, we used senescence-accelerated mouse prone 8 (SAMP8) mice to determine whether HHQ consumption increases oxidative-stress-induced injury, because in SAMP8 mice, the activity of 8-oxoguanine DNA glycosylase 1, which repairs oxidative modifications in DNA, is decreased. The results showed that two out of twelve (16.7%) HHQ-treated mice presented polyuria and glucosuria around 2 months after the start of treatment, indicating that HHQ may act as a mutagen against SAMP8 mice, which is sensitive to oxidative damage. No abnormalities were observed in the chlorogenic acid (coffee polyphenol, CPP)-treated group. The concentration of hydrogen peroxide in the serum of SAMP8 mice was significantly higher than that in SAMR1 (senescence-resistant) control mice, and the concentration was further increased in the HHQ-treated group. CPP, when coexisting with HHQ at the rate contained in roasted coffee, decreased the amount of hydrogen peroxide in the serum of SAMP8 mice. Although CPP can act both oxidatively and antioxidatively as a polyphenol, CPP acts more antioxidatively when coexisting with HHQ. Thus, the oxidative effect of HHQ was shown to be counteracted by CPP.

Emerging Roles of DNA Glycosylases and the Base Excision Repair Pathway.

The base excision repair (BER) pathway historically has been associated with maintaining genome integrity by eliminating nucleobases with small chemical modifications. In the past several years, however, BER was found to play additional roles in genome maintenance and metabolism, including sequence-specific restriction modification and repair of bulky adducts and interstrand crosslinks. Central to this expanded biological utility are specialized DNA glycosylases - enzymes that selectively excise damaged, modified, or mismatched nucleobases. In this review we discuss the newly identified roles of the BER pathway and examine the structural and mechanistic features of the DNA glycosylases that enable these functions.

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Endonuclease VIII-like 1 deficiency potentiates nigrostriatal dopaminergic neuron degeneration in a male mouse model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder caused by a characteristic loss of dopaminergic neurons in the substantia nigra and degeneration of dopamine terminals in the dorsal striatum. Previous studies have suggested that oxidative stress-induced DNA damage may be involved in PD pathogenesis, as steady-state levels of several types of oxidized nucleobases were shown to be elevated in PD brain tissues. These DNA lesions are normally removed from the genome by base excision repair, which is initiated by DNA glycosylase enzymes such as endonuclease VIII-like 1 (Neil1). In this study, we show that Neil1 plays an important role in limiting oxidative stress-induced degeneration of dopaminergic neurons. In particular, Neil1-deficient male mice exhibited enhanced sensitivity to nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and Neil1-deficient animals had higher levels of γH2AX-marked DNA damage than wild-type (WT) controls, regardless of treatment status. Moreover, MPTP-treated Neil1-/- male mice had slightly elevated expression of genes related to the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant pathway. Treatment with the Nrf2 activator, monomethyl fumarate, reduced PD-like behaviors and pathology in Neil1-/- male mice, suggesting that Neil1 is an important defense molecule in an oxidative cellular environment. Taken together, these results suggest that Neil1 DNA glycosylase may play an important role in limiting oxidative stress-mediated PD pathogenesis.

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana.

BACKGROUND: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. RESULTS: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. CONCLUSIONS: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins.

BACKGROUND: In spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host's capsule. RESULTS: The TP84_26 'capsule depolymerase' (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC-MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion. CONCLUSIONS: The novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.