Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein–protein interactions. In order to isolate the protein–DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1–VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1–VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1.     

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.     

Isolation, purification, and identification of virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.     

The carboxy-terminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded type IV transport system

Agrobacterium tumefaciens transfers DNA from the resident 'tumour-inducing' (Ti) plasmid into plant cells, where it can be stably integrated into the plant genome, ultimately resulting in crown gall tumour formation. The mobilized DNA molecule is a single-stranded intermediate with VirD2 covalently bound to its 5' end. Successful transport of the transferred DNA (T-DNA) and integration of the DNA into the genome requires that additional proteins be transported to the plant as well, including the single-stranded (ss)DNA-binding protein, VirE2. The transport of these two different substrates occurs as a result of the activities of a type IV secretion system encoded by the virB operon. Although the substrates have been identified, the mechanism of their transport remains unknown. In the experiments described here, a region in one of these substrates, VirE2, necessary for transport is identified. The addition of a C-terminal FLAG epitope tag to VirE2, or the deletion of its C-terminal 18 amino acids, renders it non-functional in A. tumefaciens. However, transgenic plants expressing either of these virE2 genes respond to virE2 mutants of A. tumefaciens by forming wild-type tumours. These results indicate that this region of VirE2 is necessary for the protein to be transported into the plant cells, but is not necessary for its function within the plant. Additionally, these studies demonstrate that mutant forms of VirE2 lacking this region do not disrupt the activities of the VirB transporter and support the hypothesis that VirE2 and the VirD2 T-strand are transported independently, even when they co-exist in the same cell.     

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.     

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.     

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.     

The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.     

Mutagenesis of the Agrobacterium VirE2 Single-Stranded DNA-Binding Protein Identifies Regions Required for Self-Association and Interaction with VirE1 and a Permissive Site for Hybrid Protein Construction

The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation. By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.     

Isolation,purification, and identification of the virulence protein VirE2 from <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through the membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of the virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, the virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein, with six histidine residues at the N-end, was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2, which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 92–98.Original Russian Text Copyright © 2005 by Volokhina, Sazonova, Velikov, Chumakov.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.     

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.     

Identification, cloning and expression of p25, an AT-rich DNA-binding protein from the extreme thermophile, Thermus aquaticus YT-1

Although the G+C content of Thermus aquaticus YT-1 chromosomal DNA is 67.4%, regions with lower G+C content have also been observed. AT-rich DNA-binding proteins may contribute to the thermostability and biological functions of these DNA regions at Thermus growth temperatures. Using double-stranded DNA (dsDNA)-cellulose chromatography, a T.aquaticus YT-1 protein, designated as p25, was identified to bind preferentially to AT-rich DNA. The gene encoding p25 was cloned and sequenced after immunoscreening T.aquaticus YT-1 expression libraries. The deduced primary structure of p25 is 211 amino acids in length with a molecular weight of 23 225 Da. Native p25 was purified and characterized as a homodimer with modification possibly at lysine and arginine residues. Its preferential and temperature-dependent binding to AT-rich DNA was confirmed with mobility-shift DNA-binding assays. The protein was demonstrated to bind preferentially to dsDNA instead of single-stranded DNA. The binding of p25 to dsDNA also improved the thermotolerence of this protein. Overexpression study of fusion p25 suggested that the N-terminus of the protein might form the DNA-binding domain or be closely involved in DNA-binding activity.     

Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.     

Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.

Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene. Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562. Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative. Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA. The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA. In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively. Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25. Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.     

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.     

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.