Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor

Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3′–5′ exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.     

Crystal structure of a metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis: Computational prediction and experimental validation of phosphoesterase activity

The crystal structures of an unliganded and adenosine 5′‐monophosphate (AMP) bound, metal‐dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a polymerase and histidinol phosphatase (PHP, Pfam: PF02811) domain with a second domain (residues 105‐178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain are unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. Theoretical microscopic anomalous titration curve shapes, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA‐proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Phosphoesterase domains associated with DNA polymerases of diverse origins.

Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members.     

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.

In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation. Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease     

Novel 3'-ribonuclease and 3'-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D

Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.     

Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D

DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.     

Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD)

DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3′-phosphomonoesterase and 3′-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues.     

Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii

Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.     

Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis

A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3'-5' exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.     

Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3

The B-subunit of Shiga toxin has been demonstrated as a powerful vector for carrying attached peptides into cells for intracellular transport studies and for medical research. We have investigated the structure of the B-subunit and of a chimera bearing a peptide extension, bound to the membranous lipidic receptor, the globotriaosylceramide (Gb3). Two-dimensional crystals of both B-subunits have been obtained by the lipid layer method and projection maps have been calculated at 8.5A resolution from ice-embedded samples. The B-subunits as the chimera are organized in a pentameric form similar to the X-ray structure of the B-subunit not bound to Gb3. A difference map of both proteins has been calculated in which no density could be attributed to the peptide extension. Cross-correlations with projections of the B-subunit X-ray structure revealed that pentamers in the 2D crystals were oriented with their binding sites pointing to the lipid layer. Thus, it is likely that the peptide extension was disordered and confined to the surface of the pentamer opposite to the Gb3 binding sites. This location confirms the hypothesis that addition of peptide extension to the C-terminus conserves the ability of the modified B-subunit to bind the membranous receptor Gb3.     

The nuclease domain of the Escherichia coli RecBCD enzyme catalyzes degradation of linear and circular single-stranded and double-stranded DNA

The 30 kDa C-terminal domain of the RecB protein (RecB30) has nuclease activity and is believed to be responsible for the nucleolytic activities of the RecBCD enzyme. However, the RecB30 protein, studied as a histidine-tagged fusion protein, appeared to have very low nucleolytic activity on single-stranded (ss) DNA [Zhang, X. J., and Julin, D. A. (1999) Nucleic Acids Res. 27, 4200-4207], which raised the question of whether RecB30 was indeed the sole nuclease domain of RecBCD. Here, we have purified the RecB30 protein without a fusion tag. We report that RecB30 efficiently degrades both linear and circular single- and double-stranded (ds) DNA. The endonucleolytic cleavage of circular dsDNA is consistent with the fact that RecB30 has amino acid sequence similarity to some restriction endonucleases. However, endonuclease activity on dsDNA had never been seen before for RecBCD or any fragments of RecBCD. Kinetic analysis indicates that RecB30 is at least as active as RecBCD on the ssDNA substrates. These results provide direct evidence that RecB30 is the universal nuclease domain of RecBCD. The fact that the RecB30 nuclease domain alone has high intrinsic nuclease activity and can cleave dsDNA endonucleolytically suggests that the nuclease activity of RecB30 is modulated when it is part of the RecBCD holoenzyme. A new model has been proposed to explain the regulation of the RecB30 nuclease in RecBCD.     

The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo

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

Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii

Functions of the terminal domains of the family D DNA polymerase from Pyrococcus horikoshii (PolDPho) were analyzed by making and characterizing various truncated proteins. Based on a co-expression vector developed previously (Shen, Y., Musti, K., Hiramoto, M., Kikuchi, H., Kawabayashi, Y., and Matsui, I. (2001) J. Biol. Chem. 276, 27376-27383), 25 vectors for terminal truncated proteins were constructed. The expressed proteins were characterized in terms of thermostability, subunit interaction, and polymerization and 3'-5' exonuclease activities. The carboxyl-terminal (1255-1332) of the large subunit (DP2Pho) and two regions, the 201-260 and 599-622, of the small subunit (DP1Pho) were found to be critical for the complex formation, and probable subunit interaction of PolDPho. The amino-terminal (1-300) of DP2Pho is essential for the folding of PolDPho and is likely the oligomerization domain of PolDPho. A short region at the extreme C-terminal of DP2Pho (from 1385 to 1434) and the N-terminal of DP1Pho(1-200), which forms a stable protein, are not absolutely necessary for either polymerization or the 3'-5' exonuclease activity. We identified a possible regulatory role of DP1Pho(1-200) for the 3'-5' exonuclease. Deletion of DP1Pho(1-200) increased the exonuclease and DNA binding activities of PolDPho. Adding DP1Pho(1-200) to the truncated protein suppressed the elevated exonuclease activity. We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3'-5' exonuclease, but is dispensable for the DNA polymerization.     

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes

There is growing recognition that white matter pathology is a common feature in Alzheimer's disease. We have previously reported that the amyloid beta peptide (Abeta) induces apoptosis in oligodendrocytes (OLG), via activation of neutral sphingomyelinase (nSMase) and resultant generation of ceramide. In the current study, we report that both Abeta and ceramide increased expression of the proapoptotic protein DP5/Hrk (DP5), and release of cytochrome C from mitochondria to cytoplasm in OLGs. We provide evidence that the Jun N-terminal kinase (JNK) signaling pathway mediates Abeta- and ceramide-induced apoptosis: Both Abeta and ceramide activated JNK phosphorylation, and subsequent AP-1 DNA binding activity; JNK siRNA decreased AP-1 DNA binding, DP5 expression and reduced cell death. Furthermore, inhibition of nSMase attenuated Abeta-induced JNK phosphorylation, AP-1 DNA binding activity, DP5 expression, and cytochrome C release. Collectively, these results suggest that Abeta-induced apoptosis involves the sequential activation of nSMase with ceramide generation, JNK activation, AP-1 DNA binding, and DP5 expression.     

Inhibition of Escherichia coli heat-labile enterotoxin B subunit pentamer (EtxB5) assembly in vitro using monoclonal antibodies

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.     

The DNA binding domain of a papillomavirus E2 protein programs a chimeric nuclease to cleave integrated human papillomavirus DNA in HeLa cervical carcinoma cells

Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.