Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites

We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites.     

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.     

Changing of filamentation dynamics of RecA protein, induced by D112R amino acid substitution or ATP to dATP replacement, results in filament steadiness TO THE RecX protein action

It is known that RecX is a negative regulator of RecA protein. We found that the mutant RecA D112R protein exhibits increased resistance to RecX protein comparatively to wild-type RecA protein in vitro and in vivo. Using molecular modeling we showed, that amino acid located in position 112 can not approach RecX closer than 25-28 angstroms. Thus, direct contact between amino acid and RecX is impossible. RecA D112R protein more actively competes with SSB protein for the binding sites on ssDNA and, therefore, differs from the wild-type RecA protein by dynamics of filamentation on ssDNA. On the other hand, after the replacement of ATP by dATP, the wild-type RecA protein, changing the dynamics of filamentation on ssDNA, also becomes more resistant to RecX. Based on these data it is concluded that the dynamics of filamentation has a great, if not dominant role in the stability of RecA filament to RecX relative to the role of RecA-RecX protein-protein interactions discussed earlier. We also propose an improved model of regulation of RecA by RecX protein, where RecA filament elongation along ssDNA is blocked by RecX protein on the ssDNA region, located outside the filament.     

ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.     

Evolutionary conservation of RecA genes in relation to protein structure and function.

Functional and structural regions inferred from the Escherichia coli R ecA protein crystal structure and mutation studies are evaluated in terms of evolutionary conservation across 63 RecA eubacterial sequences. Two paramount segments invariant in specific amino acids correspond to the ATP-binding A site and the functionally unassigned segment from residues 145 to 149 immediately carboxyl to the ATP hydrolysis B site. Not only are residues 145 to 149 conserved individually, but also all three-dimensional structural neighbors of these residues are invariant, strongly attesting to the functional or structural importance of this segment. The conservation of charged residues at the monomer-monomer interface, emphasizing basic residues on one surface and acidic residues on the other, suggests that RecA monomer polymerization is substantially mediated by electrostatic interactions. Different patterns of conservation also allow determination of regions proposed to interact with DNA, of LexA binding sites, and of filament-filament contact regions. Amino acid conservation is also compared with activities and properties of certain RecA protein mutants. Arginine 243 and its strongly cationic structural environment are proposed as the major site of competition for DNA and LexA binding to RecA. The conserved acidic and glycine residues of the disordered loop L1 and its proximity to the RecA acidic monomer interface suggest its involvement in monomer-monomer interactions rather than DNA binding. The conservation of various RecA positions and regions suggests a model for RecA-double-stranded DNA interaction and other functional and structural assignments.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Site-directed mutagenesis in the Escherichia coli recA gene

Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.     

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.     

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R ( recA2201 ) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo . We have combined the K72R variant of RecA with another mutation, RecA E38K ( recA730 ). In vitro , the double mutant RecA E38K/K72R ( recA730,2201 ) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro .     

Isolation and characterization of human HSP70 expressed in Escherichia coli

A plasmid containing the human HSP70 gene was used to transfect and express the protein in Escherichia coli. The bacterial product was a fusion protein containing 640 amino acids of HSP70, plus 33 additional NH2 terminal amino acids; 12 from the bacterial expression vector and 21 from a 5' human sequence that is not normally translated. It was partially purified by ion-exchange and ATP-Sepharose affinity column chromatography. The bacterially produced human HSP70 protein was then compared with HSP70 obtained from cultured 293 cells. Both shared the same staphylococcal V8 protease peptide fragment pattern, ATP binding, and a weak ATPase activity (about 10-15 nmol ATP hydrolyzed per milligram protein per minute at 30 degrees C). The bacterially produced human HSP70 protein differed in its V8 protease pattern with an E. coli ATP-binding protein that corresponded in molecular mass to the E. coli dnaK gene product. Mutants in the human HSP70 gene were constructed which significantly reduced a predicted major alpha-helical domain in the HSP70 molecule that has partial homology to an ATP-binding site of several protein kinases. One HSP70 mutant clone contained a deletion of 20% at the NH2 terminus, and expressed a 57-kDa product, while the other was missing the middle 50% of the gene (40-kDa product). Neither protein fragment bound to an ATP affinity column, suggesting that ATP binding to HSP70 may be conformationally affected by a region about 20% internal to the NH2 terminal end of the molecule. Recently, a similar location of the ATP-binding site has been reported by Milarski and Morimoto (27).     

Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein

Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that serine and asparagine are functionally interchangeable at position 240. The S240K mutation, in contrast, essentially eliminated the ability of the RecA protein to utilize ATP as a nucleotide cofactor. The [S240K]RecA protein was able to catalyze the hydrolysis of dATP, however, suggesting that the absence of the 2'-hydroxyl group reduced an inhibitory interaction with the Lys240 side chain. Interestingly, the [S240K]RecA protein was able to promote an efficient LexA cleavage reaction but exhibited no strand exchange activity when dATP was provided as the nucleotide cofactor. This apparent separation of function may be attributable to the elevated S(0.5) value for dATP for the [S240K]RecA protein (490 μM, compared to 20-30 μM for the wild type and [S240N]RecA proteins), and may reflect a differential dependence of the LexA co-protease and DNA strand exchange activities on the nucleotide cofactor-mediated stabilization of the functionally-active state of the RecA-ssDNA complex.     

A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryarchaeal counterparts. Rather than comprising four DNA-binding domains and a zinc-finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA-binding domain in a polypeptide of just 148 amino acids with a eubacterial-like acidic C-terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein,despite possessing DNA-binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt)per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein-mediated DNA strand-exchange and rescuing the ssb-1 lethal mutation of E. coli respectively. We discuss possible evolutionary relationships amongst the various members of the SSB/RPA family.     

C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs.

RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.     

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.     

Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation

The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.     

Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.     

Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein

The gene that codes for the surface antigen of Plasmodium knowlesi sporozoites (CS protein) is unsplit and present in the genome in only one copy. The CS protein, as deduced from DNA sequence analysis of the structural gene, has an unusual structure with the central 40% of the polypeptide chain present as 12 tandemly repeated amino acid peptide units flanked by regions of highly charged amino acids. The protein has an amino-terminal hydrophobic amino acid signal sequence and a hydrophobic carboxy-terminal anchor sequence. The coding sequence of the gene has an AT content of 53%, compared with 70% AT in the 5′ and 3′ flanking sequences, and is contained entirely within an 11 kb Eco RI genomic DNA fragment. This genomic fragment expresses the CS protein in E. coli, indicating that the parasite promoter and ribosome binding site signals can be recognized in E. coli.