DNA binding and helicase domains of the Escherichia coli recombination protein RecG

The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination. The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site. However, the regions of RecG which confer junction DNA specificity are unknown. To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes. Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus. In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding. These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.     

Viability and preliminary in vivo characterization of site-directed mutants of Escherichia coli single-stranded DNA-binding protein

Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb+ control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.     

Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme

We have explored how IL-15 influences Th1 or Th2 type immune response in vivo. Intraperitoneal application of an IL-15-IgG2b fusion protein (FP) to mice did neither significantly affect the footpad swelling nor the production of hemagglutinizing antibodies in a delayed type hypersensitivity reaction to sheep red blood cells. In contrast, in an established murine Th2 model of sensitization to ovalbumin (OVA), IL-15-IgG2b FP plus OVA sensitization resulted in massively accelerated and enhanced allergen-specific IgE and IgG1 antibody production. In vitro, stimulation of spleen cells from OVA-sensitized mice with OVA+IL-15 or OVA+IL-15-IgG2b resulted in a significantly enhanced IgE production. IL-4 secretion was significantly induced by IL-15 but not by IL-15-IgG2b. An IL-2-IgG2b FP with the same Fc tail as the IL-15-IgG2b FP was used as control in both models. In striking contrast to the IL-15-IgG2b FP, IL-2-IgG2b significantly inhibited the Th2 type antibody production in vivo. The current study suggests that IL-15-IgG2b may be employed as a potent accelerator and enhancer of Th2 type immune responses in vivo, while IL-2-IgG2b can suppress the latter.     

DNA strand invasion promoted by Escherichia coli RecT protein

The RecT protein of Escherichia coli is a DNA-pairing protein required for the RecA-independent recombination events promoted by the RecE pathway. The RecT protein was found to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the absence of Mg2+. In the presence of Mg2+, RecT binding to dsDNA was inhibited drastically, whereas binding to ssDNA was inhibited only to a small extent. RecT promoted the transfer of a single-stranded oligonucleotide into a supercoiled homologous duplex to form a D (displacement)-loop. D-loop formation occurred in the absence of Mg2+ and at 1 mM Mg2+ but was inhibited by increasing concentrations of Mg2+ and did not require a high energy cofactor. Strand transfer was mediated by a RecT-ssDNA nucleoprotein complex reacting with a naked duplex DNA and was prevented by the formation of RecT-dsDNA nucleoprotein complexes. Finally, RecT mediated the formation of joint molecules between a supercoiled DNA and a linear dsDNA substrate with homologous 3'-single-stranded tails. Together these results indicate that RecT is not a helix-destabilizing protein promoting a reannealing reaction but rather is a novel type of pairing protein capable of promoting recombination by a DNA strand invasion mechanism. These results are consistent with the observation that RecE (exonuclease VIII) and RecT can promote RecA-independent double-strand break repair in E. coli.     

The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein

Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein.     

Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase

To elucidate the role of the two conserved cis-proline residues of aspartate aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195A in which the two prolines were replaced by alanine. The crystal structures of P195A and P138A/P195A have been determined at 2.3-2.1 A resolution. The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, whereas the trans conformation is adopted at the 137-138 peptide bond. Quite surprisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein. All the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913]. The kinetics of renaturation of P195A, where the alanine retains the wild-type cis conformation, is faster than wild type, whereas renaturation of P138A, which adopts the trans conformation, is slower. We conclude that cis-prolines seem to have been retained throughout the evolution of aspartate aminotransferase to possibly play a subtle role in directing the traffic of intermediates toward the unique structure of the native state, rather than to respond to the needs for a specific catalytic or functional role.     

Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.     

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.     

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.     

Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli Rep helicase dimer

Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.     

Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates

Excision of uracil from tetraloop hairpins and single stranded ('unstructured') oligodeoxyribonucleotides by Escherichia coli uracil DNA glycosylase has been investigated. We show that, compared with a single stranded reference substrate, uracil from the first, second, third and the fourth positions of the loops is excised with highly variable efficiencies of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of E.coli single stranded DNA binding protein (SSB) in the reactions resulted in approximately 7-140-fold increase in the efficiency of uracil excision from the first, second or the third position in the loop but showed no significant effect on its excision from the fourth position. In contrast, the presence of SSB decreased uracil excision from the single stranded ('unstructured') substrates approximately 2-3-fold. The kinetic studies show that the increased efficiency of uracil release from the first, second and the third positions of the tetraloops is due to a combination of both the improved substrate binding and a large increase in the catalytic rates. On the other hand, the decreased efficiency of uracil release from the single stranded substrates ('unstructured') is mostly due to the lowering of the catalytic rates. Chemical probing with KMnO4showed that the presence of SSB resulted in the reduction of cleavage of the nucleotides in the vicinity of dUMP residue in single stranded substrates but their increased susceptibility in the hairpin substrates. We discuss these results to propose that excision of uracil from DNA-SSB complexes by uracil DNA glycosylase involves base flipping. The use of SSB in the various applications of uracil DNA glycosylase is also discussed.     

Differential proximity probing of two DNA binding sites in the Escherichia coli recA protein using photo-cross-linking methods

The DNA strand-exchange reaction catalyzed by the Escherichia coli RecA protein occurs between the two DNA binding sites that are functionally distinct. Site I is the site to which a DNA molecule (normally single-stranded DNA) binds first; this first binding makes site II available for additional DNA-binding (normally double- stranded DNA). Photo-cross linking was employed to identify the amino acid residues located close to the bound DNA molecule(s). A ssDNA oligo containing multiple 5-iodouracil residues (IdU) was cross-linked to RecA by irradiation with a XeC1 pulse laser (308 nm), and the cross-linked peptides were purified and sequenced. To differentiate the two DNA binding sites, we used two protocols for making RecA-ssDNA complexes: (1) IdU-containing oligo was mixed with a stoichiometric excess of RecA, a condition which favors the binding of the oligo to site I, and (2) RecA was first allowed to bind to a nonphotoreactive oligo and then chased with the IdU-containing oligo, a condition which favors the binding of the IdU-oligo to site II. We observed that when RecA was in excess (site I probing), cross-linking occurred to Met-164 which is located in the disordered loop 1 of the RecA crystal structure [Story, R.M., Weber, I.T., & Steitz, T.A. (1992) Nature 355, 318-325]. When site II was probed, the majority of cross-linking occurred to Met-202 or Phe-203, located in loop 2. These results support the idea that, as predicted by Story and co-workers (1992), the disordered loops are involved in DNA binding. The results also suggest that the two sites are not only functionally but also physically distinct.     

Sequence-specific binding protein of single-stranded and unimolecular quadruplex telomeric DNA from rat hepatocytes

A rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5'-d(TTAGGG)n-3'. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex copyright 200 gel filtration disclosed for uqTBP25 subunit and native Mr values of 25.4 +/- 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5'-d(TTAGGG)4-3', respectively, had dissociation constants, Kd, of 2.2 or 13.4 nM. Relative to d(TTAGGG)4, complexes with 5'-r(UUAGGG)4-3', blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher Kd values. Single base alterations within the d(TTAGGG) repeat increased the Kd of complexes with uqTBP25 by 9-215-fold. Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions.     

Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO

The effect of gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca2+]i in a dose-dependent manner. The GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na+/K+/2Cl- cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl- pump, also inhibited the GABA-induced increased in [Ca2+]i. These results suggest a role for Cl- transport processes in the GABA response. The coapplication of GABA and high K+ led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca2+ channel blocker, and abolished by the absence of extracellular Ca2+. Results indicate that the GABA response shares a common pathway of Ca2+ movement with the high K(+)-induced response. These observations suggest that the stimulation with GABA results in Ca2+ influx through voltage-gated Ca2+ channels, and that these effects are dependent on Cl- transport systems.     

Transfection of Escherichia coli by Mu DNA

The total amount of noradrenaline (NA) in the male accessories increases with normal growth of the organs and also with the age of the rat. The ductus deferens of the old rat has about twice as high NA concentration as that of the young rat. Castration of the prepuberal rat or the puberal rat leads to retardation or cessation, respectively, of the increase in NA amount of the male genital tract. Only in the old rat does castration produce a definite decrease in NA amount of the tract. Castration always raises the NA concentration of the sex accessories. Testosterone treatment of the puberal rat or the old rat produces minor or no increase, respectively, in total amount of NA of the male sex accessories. Testosterone treatment of old prepuberally castrated rats produces a marked increase in NA content of the male organs. It is concluded that androgens have no substancial direct effect on the adrenergic innervation per se, but affects the transmitter levels of the male organs indirectly through changes in number, size and relative proportion of the target cells of the adrenergic nerves i.e. the smooth muscle cells.     

Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP)

HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.     

Ndd, the bacteriophage T4 protein that disrupts the Escherichia coli nucleoid, has a DNA binding activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.