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The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination. DOI: http://dx.doi.org/10.7554/eLife.14294.001, eLife digest DNA encodes information that cells need to create the molecules and proteins that are essential for life. It is therefore vital that damaged DNA is repaired rapidly and accurately. Some DNA-damaging agents, such as gamma radiation, break both strands of the DNA double helix, which can be fatal to cells if not repaired quickly and accurately. One important pathway in charge of repairing such double-strand breaks is called the homologous recombination repair pathway. The first stage of this repair involves cutting away part of one of the DNA strands at the break. This exposes a single-stranded stretch of the partner strand, which can be used for the repair. One organism that is highly resistant to having its DNA damaged by radiation is the bacterium Deinococcus radiodurans. In this bacterium, an enzyme called RecJ performs part of the first step in the repair of DNA double-strand breaks by progressively shortening one end of a DNA strand. Cheng et al. have now used crystallography to look at the structure that RecJ forms when it binds to DNA. This, together with the results from biochemical experiments, revealed how RecJ recognizes where to bind on a broken DNA strand and how it moves along the broken strand along with cutting that strand. Further investigations revealed that two other proteins enhance the ability of RecJ to process the ends of broken DNA strands. Cheng et al. also examined the structure that RecJ forms with one of these additional proteins, called SSB. A future goal is to determine how all three proteins co-ordinate with each other to efficiently and accurately repair double stranded breaks in the D. radiodurans bacteria. DOI: http://dx.doi.org/10.7554/eLife.14294.002

The nuclease NurA is present in all known thermophilic archaea and has been implicated to facilitate efficient DNA double-strand break end processing in Mre11/Rad50-mediated homologous recombinational repair. To understand the structural and functional relationship of this enzyme, we constructed five site-directed mutants of NurA from Sulfolobus tokodaii (StoNurA), D56A, E114A, D131A, Y291A, and H299A, at the conserved motifs, and four terminal deletion mutants, StoNurAΔN (19-331), StoNurAΔNΔC (19-303), StoNurAΔC (1-281), and StoNurAΔC (1-303), and characterized the proteins biochemically. We found that mutation at the acidic residue, D56, E114, D131, or at the basic residue, H299, abolishes the nuclease activity, while mutation at the aromatic residue Y291 only impairs the activity. Interestingly, by chemical cross-linking assay, we found that the mutant Y291A is unable to form stable dimer. Additionally, we demonstrated that deletion of the C-terminal amino acid residues 304-331 of StoNurA results in loss of the physical and functional interaction with the single-stranded DNA-binding protein (StoSSB). These results established that the C-terminal conserved aromatic residue Y291 is involved in dimer formation and the C-terminal residues 304-331 of NurA are involved in the interaction with single-stranded DNA-binding protein.

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1–TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858–69. ©2015 AACR.

ICP8, the herpes simplex virus type-1 encoded single-strand DNA (ssDNA)-binding protein, promotes the assimilation of a single-stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoprotein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts single-stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52.

Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis.

We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by.E.coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming substrates with reactive groups attached to the 5' gamma-phosphate on different length "arms", we have demonstrated that, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linked to both primase and SSB could be equally utilized as initiating nucleotide for pRNA synthesis. The distance between SSB surface and alpha-phosphorus of the priming substrate was estimated to be less than 7 A. ATP cross-linked to primase and SSB can be further elongated in the presence of other NTPs, giving almost identical patterns of covalently attached pRNAs of up to 12 nucleotides in length. The regions of primase and SSB with cross-linked ATP that can be used for pRNA synthesis are, therefore, arranged in a similar way relative to the active center of pRNA synthesis. The pRNA covalently linked to SSB was localized, mapping between Met48 and Trp88. This observation raises the possibility that SSB may play an active role in the initiation of pRNA synthesis in this system.

The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.

We have identified a protein in Chlamydomonas reinhardtii cell extracts that specifically binds the single-stranded (ss) Chlamydomonas G-strand telomere sequence (TTTTAGGG)n. This protein, called G-strand binding protein (GBP), binds DNA with two or more ss TTTTAGGG repeats. A single polypeptide (M(r) 34 kDa) in Chlamydomonas extracts binds (TTTTAGGG)n, and a cDNA encoding this G-strand binding protein was identified by its expression of a G-strand binding activity. The cDNA (GBP1) sequence predicts a protein product (Gbp1p) that includes two domains with extensive homology to RNA recognition motifs (RRMs) and a region rich in glycine, alanine and arginine. Antibody raised against a peptide within Gbp1p reacted with both the 34 kDa polypeptide and bound G-strand DNA-protein complexes in gel retardation assays, indicating that GBP1 encodes GBP. Unlike vertebrate heteronuclear ribonucleoproteins, GBP does not bind the cognate telomere RNA sequence UUUUAGGG in gel retardation, North-Western or competition assays. Thus, GBP is a new type of candidate telomere binding protein that binds, in vitro, to ss G-strand telomere DNA, the primer for telomerase, and has domains that have homology to RNA binding domains in other proteins.

We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5′-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid . DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner. A five-base direct repeat is important for the DNA binding of RCS2. South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx. 43 kDa.

Escherichia coli SSB tetramers can bind to single stranded (ss) DNA in several binding modes. At 25 degrees C, pH 8.1, SSB can form at least three distinct binding modes, (SSB)n, where the number of nucleotides occluded per tetramer (n), can have values of 35, 56 or 65. Stability of the different modes is modulated by solution conditions, primarily the salt concentration and type, as well as the free SSB concentration. At least two different types of positive co-operative binding of SSB to ssDNA have also been observed, which appear to be correlated with different SSB binding modes. The (SSB)65 mode, which dominates at monovalent salt concentrations > 0.2 M, displays only moderate, "limited" co-operative binding in which clustering of SSB is limited to the formation of dimers of tetramers (octamers). However, at lower salt concentrations, "unlimited" co-operative binding is observed in which long SSB clusters can form, similar to the behavior observed for the phage T4 gene 32 protein. It has been proposed that unlimited co-operativity is linked to the (SSB)35 binding mode; however, this has not been verified since quantitative estimates of the co-operativity in this binding mode are difficult on long ssDNA. To estimate the nearest-neighbor co-operativity parameter in the (SSB)35 mode, we have examined the equilibrium binding of SSB to the oligodeoxynucleotide, dA(pA)69. Under certain conditions, 1:1 complexes, in which all four SSB subunits interact with the dA(pA)69, can form at low SSB binding densities, whereas 2:1 complexes, in which both SSB tetramers bind to DNA using only two subunits, can form at high SSB binding densities. These 2:1 complexes serve as a model for co-operative binding in the (SSB)35 binding mode. We show that SSB tetramers bind in this mode with a minimum nearest-neighbor co-operativity parameter of omega 35 = 1.0 x 10(5) (0.125 M NaCl, pH 8.1, 25 degrees C). This indicates that the nearest-neighbor co-operativities for SSB tetramers bound to ssDNA in the (SSB)35 versus the (SSB)65 mode differ qualitatively and quantitatively and suggests that the (SSB)35 mode is responsible for the ability of SSB protein to form long clusters on ssDNA. If the ability of helix destabilizing proteins to form uninterrupted protein clusters on ssDNA is important in DNA replication, then it is likely that SSB uses its (SSB)35 mode to function in this capacity.

The gene V protein of bacteriophage f1 binds to single-stranded nucleic acids and is essential for propagation of phage f1. We tested the function of gene V protein mutants with single amino acid substitutions in two ways: by the ability of the mutant proteins to support phage growth, and by the ability of the mutant proteins, when expressed at high levels, to inhibit the growth of Escherichia coli . The results of the tests were used to identify sites in the protein that are relatively tolerant or intolerant to subsitution, where tolerant sites are defined as those where most substitutions do not affect the function of the protein. The two assays generally yielded similar results for the tolerance of sites to substitution. Many sites that are less than 10% exposed to the solvent are relatively intolerant of substitution, with even very conservation substitutions leading to loss of function in some cases such as Ile to Leu at residue 6. Some buried sites such as Ile47 are more tolerant, with even a substitution of Ile to Thr leading to a functional protein based on the ability of the proteins to inhibit the growth of E. coli . Some surface sites in the protein (>10% exposure to solvent) that are thought to be near the location of bound oligonucleotides, such as Arg16, Val19, Ser20, Arg21, Tyr26, Lys46 and Arg80, are sensitive to substitution. Other side-chains thought to be close to bound oligonucleotides, including Leu28, can be replaced with a number of amino acids with little loss of function based on either assay. Most non-Gly/Pro surface residues thought to be distant from the locations of bound oligonucleotides are relatively tolerant of substitution, except for two small residues (Ala11 and Thr14), two aromatic residues (Tyr34 and Tyr56), two residues that are only partially exposed to solvent (Asn29 and Val170), and three residues that have been proposed to be at the dimer-dimer interface formed when gene V protein binds to nucleic acids (Glu40, Try41 and Arg82).

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.

We have identified two regions of non-random purine/pyrimidine strand asymmetry that were nearly identical in sequence in the 5' flanking (promoter) regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene and the human MUC1 gene. These regions contain perfect mirror repeat elements, a sequence motif previously found to be associated with the formation of H-DNA conformations. In this report we demonstrate that a single-stranded non-B DNA conformation exists at low pH in supercoiled plasmids containing the similar mirror repeat elements, and that S1 nuclease digestion maps the single-stranded region to the position of the mirror repeats. In addition, we identify a nuclear protein of approximately 27 kD that binds to single-stranded oligonucleotides corresponding to the purine-rich strand of this region, but not to the pyrimidine-rich strands or to double-stranded oligonucleotides with corresponding purine/pyrimidine strand asymmetry.

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

We have used circular dichroism (CD) spectroscopy and gel electrophoresis to characterize the single-strand DNA binding protein (ssDBP) of the bacteriophage Pf3 and its complexes with Pf3 DNA and various DNA and RNA homopolymers. The secondary structure of Pf3 ssDBP had < 1% alpha-helix and therefore was probably a beta-sheet structure like the fd gene 5 protein (g5p). From CD titrations, the binding stoichiometry of Pf3 ssDBP was two nucleotides per protein monomer (n = 2) for complexes formed with all of the nucleic acids except poly[r(U)], for which n = 3 (in a buffer of 10 mM Tris-HCl and 70 mM NaCl, pH 8.2). Evidence of an additional binding mode of n = 4 for complexes formed with Pf3 DNA was found by gel electrophoresis experiments. Pf3 ssDBP showed a marked sequence dependence in binding affinities similar to that known for the fd g5p.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000. It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S. The interaction of D-SSB with ssDNA was examined by the quenching of intrinsic protein fluorescence. The binding site size was determined to be n = 22 +/- 4 nucleotides with a maximum quenching Qm = 35 +/- 3%. Equilibrium titrations indicate that D-SSB binds with low cooperativity, omega = 10-300, and high apparent affinity, K omega = (0.7-5) x 10(7) M-1, at 225 mM NaCl. Sedimentation of D-SSB bound to small oligonucleotides demonstrates that D-SSB does not require protein association for binding. D-SSB stimulates the extent and processivity of DNA synthesis of its cognate DNA polymerase alpha. On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins.

The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.

Replication protein A (RP-A) is a heterotrimeric single-stranded DNA binding protein with important functions in DNA replication, DNA repair and DNA recombination. We have found that RP-A from calf thymus can unwind DNA in the absence of ATP and MgCl2, two essential cofactors for bona fide DNA helicases (Georgaki, A., Strack, B., Podust, V. and Hübscher, U. FEBS Lett. 308, 240-244, 1992). DNA unwinding by RP-A was found to be sensitive to MgCl2, ATP, heating and freezing/thawing. Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA. DNA unwinding by RP-A did not show directionality. Experiments with monoclonal antibodies strongly suggested that the 70kDa subunit is responsible for DNA unwinding. Phosphorylation of the 32kDa subunit of RP-A by chicken cdc2 kinase facilitated DNA unwinding indicating that this posttranslational modification might be important for modulating this activity of RP-A. Finally, DNA unwinding of a primer recognition complex for DNA polymerase delta which is composed of proliferating cell nuclear antigen, replication factor C and ATP bound to a singly-primed M13DNA slightly inhibited DNA unwinding. An important role for DNA unwinding by RP-A in processes such as initiation of DNA replication, fork propagation, DNA repair and DNA recombination is discussed.

An element within the Preprotachykinin A (PPT) promoter is highly homologous to an element from the rat type II Na channel promoter. This Na Channel element has been previously proposed to be common to a number of neuronal genes. We demonstrate that the PPT element binds a sequence specific DNA binding protein. The protein binds to only one strand of the PPT element and has little or no specificity for the double stranded DNA species. Gel retardation analysis indicates that the protein is found in both rat neuronal tissue and adult dorsal root ganglia neurons in culture but not in established tissue culture cell lines. Using the PPT element linked to magnetic beads we have been able to demonstrate the enrichment of a protein with a molecular weight of 40k with that of the binding activity. A mechanism for protein binding to the DNA is proposed based on the fact that the region binding the protein is the loop of a larger stem-loop structure in the DNA.

A CArG box motif [CC(A+T-rich)6GG] is one of the DNA elements required for muscle-specific gene transcription. Nuclear factors in mouse C2 myogenic cells strongly bind to the CArG box in the first intron of the gene (Sm alpha-A) encoding human smooth muscle alpha-actin. To clone cDNAs of the CArG box-binding factor (CBF), lambda gt11 cDNA expression libraries from C2 cells were screened for in situ DNA binding specific for this CArG box sequence. The 1.6-kb cDNA (CBF-A) encoding 285 amino acids (aa) was obtained, and a beta-galactosidase fusion protein, bacterially produced from the cDNA, bound to DNA fragments containing several CArG boxes. When the expression level of CBF-A in C2 cells increased by transfection of CBF-A expression plasmids, Sm alpha-A transcription was repressed. The deduced aa sequence of CBF-A is similar to some single-stranded (ss) nucleic acid-binding proteins. The fusion protein could bind to ssDNA, whereas CBF in C2 cell nuclear extracts could not. From these results, CBF-A is a novel CArG box-, ssDNA- and RNA-binding protein, as well as a repressive transcriptional factor.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.

The Escherichia coli single-strand binding (SSB) protein is an essential protein involved in DNA replication, recombination, and repair processes. The tetrameric protein binds to ss nucleic acids in a number of different binding modes in vitro. These modes differ in the number of nucleotides occluded per SSB tetramer and in the type and degree of cooperative complexes that are formed with ss DNA. Although it is not yet known whether only one or all of these modes function in vivo, based on the dramatically different properties of the SSB tetramer in these different ss DNA binding modes, it has been suggested that the different modes may function selectively in replication, recombination, and/or repair. The transitions between these different modes are very sensitive to solution conditions, including salt (concentration, as well as cation and anion type), pH, and temperature. We have examined the effects of multivalent cations, principally the polyamine spermine, on the SSBss poly(dT) binding mode transitions and find that the transition from the (SSB)35 to the (SSB)56 binding mode can be induced by micromolar concentrations of polyamines as well as the inorganic cation C0(NH3)2+. Furthermore, these multivalent cations, as well as Mg2+, induce the binding mode transition by binding cooperatively to the SSB-poly(dT) complexes. These observations are interesting in light of the fact that polyamines, such as spermidine, are part of the ionic environment in E. coli and hence these cations are likely to affect the distribution of SSBss DNA binding modes in vivo. Furthermore, the ability of the SSB protein to enhance the rate of renaturation of complementary single-stranded DNA >5000-fold is directly dependent upon the presence of polyamines (Christiansen, C., & Baldwin, R. L. (1977) J. Mol. Biol. 115, 4411.

Bacteriophage T4 gene 32 protein (g32P) is a DNA replication accessory protein that binds single-stranded (ss) nucleic acids nonspecifically, independent of nucleotide sequence. G32P contains 1 mol of Zn(II)/mol of protein monomer, which can be substituted with Co(II), with maintenance of the structure and activity of the molecule. The Co(II) is coordinated via approximately tetrahedral ligand symmetry by three Cys sulfur atoms and therefore exhibits intense S(-)----Co(II) ligand to metal charge-transfer (LMCT) transitions in the near ultraviolet [Giedroc, D. P., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452-8456]. A series of fluorescent 1,N6-ethenoadenosine (epsilon A)-containing oligonucleotides conforming to the structure (5'----3') d[(Tp)m epsilon A(pT)l-m-1] where 0 less than or equal to m less than or equal to l - 1 and length (l) six or eight nucleotides have been evaluated as dynamics probes and potential fluorescence energy transfer donors to Co(II) in mapping the spatial proximity of the (fixed) intrinsic metal ion and a variably positioned epsilon A-base in a series of protein-nucleic acid complexes. We provide spectroscopic evidence that the epsilon A-oligonucleotides bind to g32P-(A + B) with a fixed polarity of the phosphodiester chain. A Trp side chain(s) makes close approach to a epsilon A base positioned toward the 3' end of a bound l = 8 oligonucleotide. Six oligonucleotides of l = 8 and m = 0, 1, 3, 5, 6, or 7 were investigated as energy transfer donors to Co(II) at 0.1 M NaCl, pH 8.1, 25 degrees C upon binding to Co(II)-substituted or Zn(II) g32P-(A + B), i.e., in the presence and absence of an energy acceptor, respectively. Detectable quenching of the epsilon A-fluorescence by the Co(II)-LMCT acceptors was found to occur in all epsilon A-oligonucleotide-protein complexes, yielding energy transfer efficiencies (E) of 0.43, 0.31, 0.26, 0.26, 0.28, and 0.41 for l = 8 and m = 0, 1, 3, 5, 6, and 7 epsilon A-oligonucleotides, respectively. The two-dimensional distances R (in A) were found to vary as follows: d[epsilon A(pT)7] (m = 0), 16.0 (15.5-16.9); d[Tp epsilon A(pT)6] (m = 1), 17.7 (16.9-19.1); d[(Tp)3 epsilon A(pT)4] (m = 3), 20.7 (19.5-22.1); d[(Tp)5 epsilon A(pT)2] (m = 5), 20.5 (19.5-21.9); d[(Tp)6 epsilon ApT] (m = 6), 19.0 (18.0-20.4); and d[(Tp)7 epsilon A] (m = 7), 18.6 (17.8-19.8).(ABSTRACT TRUNCATED AT 400 WORDS)

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.

Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5′ end or a 3′ end; but in agreement with the report of Konforti &, Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5′ ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5′ to 3′ direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3′ end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5′ versus a 3′ end, we suggest that they may be processed differently in vivo .

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

Archaea have recombination proteins similar to those of eukaryote, but many have not been characterized. Here, the characterization of a Rad55 homologue from Sulfolobus tokodaii (stRad55A) was reported. StRad55A protein preferred binding to ssDNA and had ssDNA-dependent ATPase activity. In addition, UV light could induce the expression of this protein, which was different from RadB, a RadA paralog found in euryarchaeota. Most importantly, stRad55A could release the suppression of excessive stSSB (single strand DNA binding protein from S. tokodaii) on the strand exchange catalyzed by stRadA (RadA homologue from S. tokodaii), by interacting directly with both stRadA and stSSB. StRad55A may function as a mediator to accelerate the displacement of stSSB by stRadA.

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA–DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Ori s , by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Ori s . P1 nuclease sensitivity of Ori s and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Ori s sequences.

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.

The herpes simplex virus type-1 single-strand DNA-binding protein ICP8 is a 128-kDa zinc metalloprotein. In this communication we have shown that unsubstituted and bromodeoxyuridine-substituted oligonucleotides can be specifically crosslinked to ICP8 by UV irradiation. We have used this approach to show that the single-strand DNA-binding site of ICP8 resides within a 53.5-kDa tryptic polypeptide. This polypeptide initiates at alanine 368 and was estimated to extend through arginine 902. A polypeptide encompassing residues 368–902 synthesized in vitro exhibited single-strand DNA-binding activity. We conclude that the region encompassing residues 368–902 contains the single-strand DNA-binding site of ICP8. Moreover, photoaffinity labeling of ICP8 with oligonucleotides provides a means of specifically modifying its single-strand DNA-binding site, thereby facilitating future studies on the importance of its single-strand DNA-binding activity in its interaction with other DNA replication enzymes.

In previous gel-shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence-specific manner to single-stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1-DNA complex was subjected to UV irradiation to induce cross-linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel-shift assays, we were able to achieve approximately 500,000-fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV cross-linking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined.

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5′ untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns. The first exon contained only 5′ untranslated sequence. The 16-amino acid mitochondrial targeting presequence, encoded by the second and third exons, was precisely bisected by intron 2. All intron donor and acceptor sites were consistent with the GT/AG consensus. The transcription start site was determined by primer-extension analysis to be 69 bp upstream of the translation start codon. The upstream sequence lacked TATA and CCAAT boxes at the expected locations, but did contain several other potential regulatory elements including a GC box (Sp1-binding site) and three NRF-2 sites, one of which was located precisely beside the transcription start site. A 10 out of 12 imperfect NRF-1 site was located within the first exon. The 5′ flanking sequence (−546 to +30) was shown to have strong promoter activity in transient transfection assays in primary rat hepatocytes and HepG2 cells. In addition, evidence for the existence of several mtSSB processed pseudogenes was obtained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5′ upstream region of one of the pseudogenes was analyzed and found to contain negligible promoter activity.

The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively. These proteins are required for SOS mutagenesis, although their mechanism of action is unknown. By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E. coli. To examine the interaction at the protein level, the MucA, MucA′, and MucB proteins were overproduced, purified in denatured state, and refolded. Purified MucA and MucA′ each formed homodimers, whereas MucB was a monomer under native conditions. RecA promoted the cleavage of MucA to MucA′, and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins. Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins. Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide. Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB. These results suggest that MucB induces a major conformational change in the SSB·ssDNA complex but does not promote massive release of SSB from the DNA. The interaction with SSB might be related to the role of MucB in SOS-regulated mutagenesis.

Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA.

In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.

The GH receptor is essential for the actions of GH on growth and metabolism. Electromobility shift assay established that a 42-bp enhancer element in the promoter of the L1 transcript of the murine GH receptor bound nuclear proteins specific for the coding strand or the DNA duplex. Using methylation interference footprinting and electromobility shift assay with mutant oligonucleotides, the DNA-binding sites for the single-strand DNA-binding protein (SSBP) and the double-strand DNA-binding protein (DSBP) were mapped and shown to be contiguous with partial overlap. Shift-Western analysis indicated that the SSBP was a component of the DSBP complex. A functional interaction between SSBP and DSBP was suggested by the effect of the exclusion of SSBP on equilibrium binding and dissociation rate ("off rate") of the DSBP-DNA complex. Experiments using the anionic detergent deoxycholate provided evidence for a direct protein-protein interaction between SSBP and DSBP. Using lectin-affinity chromatography, discordance between the pattern of O-glycosylation of SSBP and DSBP was demonstrated. Transient transfection experiments support the role of SSBP as a repressor of DSBP's activation of transcription of the GH receptor gene. Southwestern analysis indicated that a protein of molecular mass 23-kDa exhibited binding activity specific to the coding strand of the enhancer element. We conclude that single- and double-strand DNA-binding proteins conjointly regulate the expression of the murine GH receptor gene.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: ori L and two copies of ori S . These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding protein (UL9 protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 protein is unable to unwind ori S or the box I element of ori S , either in the presence or absence of the HSV-1-encoded single-strand DNA binding protein, ICP8. However, a complex of the UL9 protein and ICP8 can unwind box I if it contains a 3′ single-stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 protein bound to box I, and ICP8 bound to single-stranded DNA generated at the A+T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses—e.g., simian virus 40 or bovine papilloma virus—in which the initiator protein, T antigen, or E1 protein alone, unwinds elements of the origin sequence, and the single-strand DNA binding protein serves only to keep the separated strands apart.

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.

A contact analysis and a series of restrained molecular dynamics simulations were employed to derive a model of the complex between single-stranded DNA and the single-stranded DNA-binding protein encoded by gene V of the filamentous phage M13. The study is based on the recently elucidated solution structure of the Tyr41→His mutant of the protein. Electron microscopy studies, indicating that the complex forms a flexible, left-handed helical coil with a diameter of 8 to 9 nm and an average pitch of 9 nm, were taken into consideration. The contact analysis served to determine the helix parameters that permit the energetically most favourable packing of protein molecules. Then a protein super-helix was built, into which two extended strands of DNA were modelled using restrained molecular dynamics. Specific constraints were included to ensure that the DNA would position itself into the binding groove of the protein. These constraints are based on recent NMR spin label experiments which offered a direct identification of the amino acids of the protein present in the DNA-binding domain. We present a model for the complex which is in full agreement with the existing reliable biophysical and biochemical data. A description of the protein-protein interface is given and the protein-DNA interaction is discussed in view of the derived model. In addition, we demonstrate that, on the basis of the available experimental data, and not imposing the left-handedness of the nucleoprotein complex, it is feasible to build also a plausible model for the complex which exhibits the opposite, i.e. right-handed, helical sense. This nucleoprotein structure features characteristics highly similar to those of the left-handed helix.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.

We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.

Bacteriophage φ29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB) which stimulates viral DNA replication. In the present study, a structural characterization of the complex between ssDNA and the φ29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of φ29 SSB upon binding. Phage φ29 SSB behaves as a monomer in solution and forms complexes with ssDNA which have a homogeneous structure, as if they consist of a continuous array of protein bound to DNA. Interaction of φ29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of φ29 SSB bound to the ssDNA and the maximal quenching upon binding was very high (Qmax = 94?6±3·5%). Direct titration experiments have allowed us to estimate that the stoichiometry (n) of binding to ssDNA was 3·4(±0·3) nucleotides per φ29 SSB monomer. Both Qmax and n are independent of the salt concentration, suggesting the existence of only one major binding mode. At low salt concentrations, the effective binding constant (Keff = Kw) to poly(dT) was 2·2 × 105 M-1 the intrinsic binding constant (K) and the cooperativity parameter (w) being 4·3 × 103 M-1 and 51, respectively. At increasing salt concentrations, the Keff exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of φ29 SSB during viral DNA replication is discussed., This investigation has been aided by Research grant from the National Institutes of Health (5 ROl GM27242-14)

The herpes simplex virus type 1 encoded ICP8 protein binds single-stranded (ss) DNA and is required for DNA replication in vitro . We have used electron microscopy to examine the ability of ICP8 to promote homologous pairing and strand transfer reactions. Visualization of M13 ssDNA-ICP8 complexes showed that they preferentially bound and enveloped homologous double-stranded (ds) DNA fragments; their deproteinization released ssDNA circles containing dsDNA segments, and an equal number of linear single strands. Optimal transfer required Mg 2+ but not nucleoside triphosphates, and showed a fourfold preference for dsDNA fragments with a few bases recessed ends, Gel electrophoretic analysis confirmed the strand transfer activity of ICP8.

We have systematically substituted the four tryptophan residues of the single-stranded DNA binding protein from Escherichia coli (EcoSSB) by polar (serine or threonine) and aromatic (tyrosine or phenylalanine) amino acids. The resulting mutants with either single amino acid exchanges or triple substitutions are all active in ssDNA binding, though in some cases with reduced affinities. Measurements of the fluorescence of the mutated EcoSSBs show that there is no interaction between the four different tryptophan residues. We analyzed the ssDNA binding of the mutant proteins by fluorescence titrations. At 0.3 M NaCl ("high salt"), all singly substituted proteins bind to poly(dT) in a manner comparable to wild-type EcoSSB, covering 65 nucleotides with 1 EcoSSB tetramer. W54S mutant protein is an exception since even at 0.3 M NaCl it covers approximately 35 nucleotides, a behavior which is typical of salt concentrations below 10 mM NaCl ("low salt"). From this observation, it is inferred that tryptophan-54 is involved in a direct interaction with the ssDNA favoring the "high-salt" binding mode. All mutant proteins lacking tryptophan-54 but possessing tryptophan-88 at "low-salt" concentrations show a nonmonotonous behavior in the fluorescence titrations. This behavior can be interpreted assuming a model of cooperative binding of EcoSSB to poly(dT) with two different binding site sizes (n approximately 27 and n approximately 33) and different binding affinities. A quantitative treatment of the problem of multiple binding modes in the interaction of a multidentate ligand with a linear polymer is applied to these titrations.

We have retro-transcribed and amplified by PCR the full-length cDNAs specifying the rat and human precursors of the single-stranded mitochondrial DNA (mtDNA)-binding protein (mtSSB). Each deduced sequence is composed of a 16-amino-acid (aa) N-terminal basic pre-sequence and a mature protein (132 aa in humans and 135 aa in the rat). The mature proteins are highly conserved among themselves and with the mtSSB from Xenopus laevis (Xl). Moreover, three regions of the protein are similar to corresponding domains of the SSB of Escherichia coli and to the E. coli F-sex factor SSB, indicating the existence of a broad class of DNA-binding proteins with structural and functional similarities both in prokaryotes and in prokaryote-derived organelles of higher organisms.