Search

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells., (Copyright © 2024. Published by Elsevier Inc.)

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS 9 ). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS 9 and TerL are functional, and that they also function with gp74. Purified TerS 9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Å resolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = β = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1., (© 2018 Elsevier Inc. All rights reserved.)

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.

We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation., (© 2015 John Wiley & Sons Ltd.)

The HNH endonuclease superfamily usually contains a conserved HNH motif in the sequence, and the second subfamily of it uses N to replace the second H in the HNH motif. A bacterium with extracellular thermostable DNase was isolated and identified as Exiguobacterium sp. yc3. A 20 kDa putative DNase was later purified and the encoding gene of it was amplified and sequenced, the deduced amino acid sequence analysis showed that the protein belongs to the HNH endonuclease superfamily, and therefore it was named as EheA ( E: xiguobacterium H: NH E: ndonuclease). Characterization of the recombinant EheA confirmed that EheA is a DNase. By site-directed mutation method, H116, N141 and N156 were proved to be essential for the DNase activity. EheA is the first experimentally determined bacterial source endonuclease belonging to the second subfamily of HNH superfamily. Further bioinformatic analysis showed that EheA homologue genes are conserved in the Exiguobacterium species, which suggests their possible important functions for Exiguobacterium species. And as a thermostable DNase, EheA also has a promising future in many application fields., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools., Results: In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice., Conclusions: A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.

Cas1 genes encode the signature protein of the CRISPR/Cas system, which is present in all CRISPR-containing organisms. Recently, Cas1 proteins (together with Cas2) have been shown to be essential for the formation of new spacers in Escherichia coli, and purified Cas1 proteins from Pseudomonas aeruginosa and E. coli have been shown to possess a metal-dependent endonuclease activity. Here we describe the protocols for the analysis of nuclease activity of purified Cas1 proteins against various DNA substrates including Holliday junctions and other intermediates of DNA recombination and repair.

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.

In response to ionizing radiation, the MRE11/RAD50/NBN complex re-distributes to the sites of DNA double-strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for the activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine-to-alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine-to-glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared with control cells, whereas blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving chromosomal DNA. EndoG is the main apoptotic endonuclease in the caspase-independent pathway. Mouse EndoG without the mitochondrial localization signal (amino-acid residues 1-43) was successfully overexpressed, purified and crystallized using a microbatch method under oil. The initial crystal (type I) was grown in the presence of the detergent CTAB and diffracted to 2.8 A resolution, with unit-cell parameters a = 72.20, b = 81.88, c = 88.66 A, beta = 97.59 degrees in a monoclinic space group. The crystal contained two monomers in the asymmetric unit, with a predicted solvent content of 46.6%. Subsequent mutation of Cys110 improved the stability of the protein significantly and produced further crystals of types II, III and IV with space groups C2, P4(1)2(1)2 (or P4(3)2(1)2) and P2(1)2(1)2(1), respectively, in various conditions. This suggests the critical involvement of this conserved cysteine residue in the crystallization process.

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.

We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protein preparation was apparently free of host cell proteins, endotoxins, protease, and aggregates. The N-terminal amino acid sequence of IL-1beta was in full agreement with the natural mature form of IL-1beta. The homogeneity of the product was further shown by X-ray structure determination which confirmed the previously solved structure of the protein. We propose the applied workflow as a strategy for industrial production of protein-based biopharmaceuticals.

Endonuclease G is a mitochondrial protein implicated in DNA fragmentation during apoptosis in cell types ranging from fungi to mammals. Features of programmed cell death have been reported in a number of single-celled organisms, including the human trypanosomatid parasites Leishmania and Trypanosoma. However, the protozoan cell death pathways and the effector molecules involved in such processes remain to be identified. In this report, we describe the pro-apoptotic function of endonuclease G in trypanosomatid parasites. Similar to metazoans, trypanosome endoG showed intrinsic nuclease activity, is localized in mitochondria and is released from this organelle when cell death is triggered. Overexpression of endoG strongly promoted apoptotic cell death under oxidant or differentiation-related stress in Leishmania and, conversely, loss of endoG expression conferred robust resistance to oxidant-induced cell death in T. brucei. These data demonstrate the conservation of the pro-apoptotic endonuclease activity of endoG in these evolutionarily ancient eukaryotic organisms. Furthermore, nuclear DNA degradation by endoG upon release from mitochondria might represent a caspase-independent cell death mechanism in trypanosomatid parasites as genes encoding caspase-like proteins have not been identified in their genomes.

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of 19 previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products.

We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

Ca(2+)-, Mg(2+)-dependent wheat endonuclease WEN1 with molecular mass of about 27 kDa was isolated from coleoptyles. Methylated DNA of lambda phage grown on E. coli dam(+), dcm(+) cells was hydrolyzed by WEN1 more effectively than DNA of phage grown on dam(-), dcm(-) cells. Two pH activity maxima (pH 6.5-7.5 and 9.0-10.5) were observed when double-stranded DNA was hydrolyzed. WEN1 is stable at elevated temperatures (65 degrees C ) and in wide range of pH values. WEN1 is activated by S-adenosyl-L-methionine, S-adenosyl-L-homocysteine and S-isobutyladenosine. It is a first case to show that higher eukaryote endonuclease discriminates between DNA of various methylation status and is modulated by S-AdoMet and its analogs.

DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.

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.

In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase delta-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5' and 3' tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5' and 3' regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.

A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5 -triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5 -monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate.

Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been studied in vitro using cell extracts or highly purified repair factors and radiolabeled DNA of known sequence with DNA damage at a defined site. This chapter describes procedures for preparation of DNA substrates designed for analysis of damage recognition, either the 5' or the 3' incision event, excision (resulting from concerted dual incisions), and repair synthesis. Excision in Escherichia coli is accomplished by the three-subunit Uvr(A)BC excision nuclease and in humans by six repair factors: XPA, RPA, XPChR23B, TFIIH, XPFERCC1, and XPG. This chapter outlines methods for expression and purification of these essential repair factors and provides protocols for performing each of the in vitro repair assays with either the E. coli or the human excision nuclease.

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2' DNA and the resulting protein-DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.

The Saccharomyces cerevisiae mutant strain YW778, which lacks apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase DNA repair activities, displays high levels of spontaneous mutations and hypersensitivities to several DNA damaging agents. We searched a cDNA library derived from the nematode Caenorhabditis elegans for gene products that would rescue the DNA repair defects of this yeast mutant. We isolated two genes, apn-1 and exo-3, encoding proteins that have not been previously characterized. Both APN-1 and EXO-3 share significant identity with the functionally established Escherichia coli AP endonucleases, endonuclease IV and exonuclease III, respectively. Strain YW778 expressing either apn-1 or exo-3 shows parental levels of spontaneous mutations, as well as resistance to DNA damaging agents that produce AP sites and DNA single strand breaks with blocked 3'-ends. Using an in vitro assay, we show that the apn-1 and exo-3 genes independently express AP endonuclease activity in the yeast mutant. We further characterize the EXO-3 protein and three of its mutated variants E68A, D190A, and H279A. The E68A variant retains both AP endonuclease and 3'-diesterase repair activities in vitro, yet severely lacks the ability to protect strain YW778 from spontaneous and drug-induced DNA lesions, suggesting that this variant E68A may possess a defect that interferes with the repair process in vivo. In contrast, D190A and H279A are completely devoid of DNA repair activities and fail to rescue the genetic instability of strain YW778. Our data strongly suggest that EXO-3 and APN-1 are enzymes possessing intrinsic AP endonuclease and 3'-diesterase activities.

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.

Two lethal factors (named plancitoxins I and II for major and minor toxins, respectively) with the same LD50 (i.v. injection into mice) of 140 microg/kg were purified from spines of the crown-of-thorns starfish Acanthaster planci. Injection of a sublethal dose of plancitoxin I or II into mice remarkably elevated serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, demonstrating that both toxins are potently hepatotoxic. Analysis by SDS-PAGE revealed that both plancitoxins are composed of two subunits (alpha-subunit of 10 kDa and beta-subunit of 27 kDa) bridged by a disulfide bond. Based on the determined N-terminal amino acid sequences of alpha- and beta-subunits, the full-length cDNA (1820 bp) encoding plancitoxin I was cloned by RT-PCR, 3'-RACE and 5'-RACE. alpha-Subunit (92 amino acid residues) and beta-subunit (240 residues) are coded in this order by the same cDNA. Interestingly, the deduced amino acid sequence of plancitoxin I showed 40-42% homologies with mammalian deoxyribonucleases II (DNases II). In addition, plancitoxin I exhibited DNA degrading activity with an optimum pH of 7.2. Plancitoxin I is the first example of toxic DNases II whose structures have been elucidated.

UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.

Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH(4))(2)SO(4) precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgCl(2) above 150 mM, MnCl(2) above 200 mM, ZnCl(2) above 150 mM, CaCl(2) above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.

Mammalian cells transfected with the S. cerevisiae APN1 protein acquire resistance to oxidizing agents, the damage of which is mainly repaired via DNA base excision repair (BER). We have recently hypothesized that this effect might be linked to the possible capacity of APN1 to accelerate mammalian BER by its 3' diesterase activity. We have investigated here the effect of pure APN1 protein on BER performed by mouse embryonic fibroblast extracts. No significant acceleration was observed in the repair of either a single AP site cleaved by the bifunctional glycosylase NTH of E. coli or the repair of a single 8-oxoguanine, initiated by the bifunctional glycosylase OGG1. Similarly, no significant effect was observed on the repair of a single U (initiated by the monofunctional glycosylase U DNA glycosylase) or the repair of a single natural abasic site. The inability of APN1 to increase the efficiency of BER initiated by bifunctional glycosylases indicates that removal of 3' blocking fragments is not the rate-limiting step of this repair pathway.

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil and hypoxanthine. In Escherichia coli two enzymes initiate repair at hypoxanthine residues in DNA. The alkylbase DNA glycosylase, AlkA, initiates repair by removal of the damaged base, whereas endonuclease V, Endo V, hydrolyses the second phosphodiester bond 3' to the lesion. We have identified and characterised a mouse cDNA with striking homology to the E.coli nfi gene, which also has significant similarities to motifs required for catalytic activity of the UvrC endonuclease. The 37-kDa mouse enzyme (mEndo V) incises the DNA strand at the second phosphodiester bond 3' to hypoxanthine- and uracil-containing nucleotides. The activity of mEndo V is elevated on single-stranded DNA substrate in vitro. Expression of the mouse protein in a DNA repair-deficient E.coli alkA nfi strain suppresses its spontaneous mutator phenotype. We suggest that mEndo V initiates an alternative excision repair pathway for hypoxanthine removal. It thus appears that mEndo V has properties overlapping the function of alkylbase DNA glycosylase (Aag) in repair of deaminated adenine, which to some extent could explain the absence of phenotypic abnormalities associated with Aag knockout in mice.

MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system. We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. Under the various conditions that we have standardized for the affinity purification of His(6) MutH, the yield of the purified MutH has been estimated to be 35-40 mg of protein from 1liter of M9 minimal media. The yield, thus, obtained by this method is significantly higher than those of previously reported methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis revealed that the protein was pure and existed essentially in a monomeric form. Uniformly 15N-labeled protein, thus, produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The dispersion seen in 15N-1H cross-peaks indicates the presence of a well-ordered structure for MutH and proper folding of the purified protein. The spectrum confirms further the existence of MutH as a monomer.

Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional column steps completed purification. The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging. Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.