DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)     

UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli

The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood. In particular, the reason for rep uvrD double mutant lethality remains obscure. We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication. Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo. In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant. In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity. We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover. This activity of UvrD is similar to that of the yeast Srs2 helicase.     

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.     

Escherichia coli Rep DNA helicase and error-prone DNA polymerase IV interact physically and functionally

Escherichia coli DNA polymerase IV, encoded by the dinB gene, is a member of the Y family of specialized DNA polymerases. Pol IV is capable of synthesizing past DNA lesions and may help to restart stalled replication forks. However, Pol IV is error-prone, contributing to both DNA damage-induced and stress-induced (adaptive) mutations. Here we demonstrate that Pol IV interacts in vitro with Rep DNA helicase and that this interaction enhances Rep's helicase activity. In addition, Pol IV polymerase activity is stimulated by interacting with Rep, and Pol IV β clamp-binding motif appears to be required for this stimulation. However, neither Rep's helicase activity nor its ability to bind DNA is required for it to stimulate Pol IV's polymerase activity. The interaction between Rep and Pol IV is biologically significant in vivo as Rep enhances Pol IV's mutagenic activity in stationary-phase cells. These data indicate a new role for Rep in contributing to Pol IV-dependent adaptive mutation. This functional interaction also provides new insight into how the cell might control or target Pol IV's mutagenic activity.     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins

The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin. Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S). The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities. When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities. This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA. This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA. The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides. Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N. & Kornberg, A. (1981) J. Biol. Chem. 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities. However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein. Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions. This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein. The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin. Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.

Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.     

Escherichia coli ribosomal protein L3 stimulates the helicase activity of the Bacillus stearothermophilus PcrA helicase.

Escherichia coli ribosomal protein L3 stimulates the in vitro helicase activity of Bacillus stearothermophilus PcrA helicase upon a variety of different substrates. L3 has no intrinsic helicase or ATPase activity nor is it able to stimulate the ATPase activity of PcrA. Gel mobility shift assays revealed that the affinity of PcrA for a variety of different DNA species (single-stranded, nicked and 3'-tailed) was enhanced in the presence of L3. We suggest that the stimulatory effect of L3 upon the helicase activity of PcrA is mediated via a protein-protein interaction which promotes cooperative binding of PcrA to its DNA substrate. This activity of L3 appears to be specific for PcrA helicase.     

Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme.

A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating. The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA. The average number of base pairs unwound per binding event (N) is sensitive to both mono- and divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl. The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a KN value for ATP of 41 +/- 9 microM and a limiting value of 32 +/- 1.8 kb/end for the number of base pairs unwound. The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.     

Identification of Escherichia coli DNA helicase IV with the use of a DNA helicase activity gel.

A DNA helicase activity gel was developed based on the assumption that DNA helicases could unwind double-stranded DNA in a polyacrylamide matrix. The production of single-stranded DNA was detected by staining the activity gel with acridine orange and visualizing the gel under long-wave UV light. The products of DNA helicase activities appeared as red bands within a green fluorescent background. A novel DNA helicase, called helicase IV, was detected in crude extracts of Escherichia coli with the use of the helicases activity gel assay. The new DNA helicase was purified to near homogeneity. The chromatographic properties and the sequence of its 11 amino-terminal residues proved that helicase IV was distinct from all of the previously described DNA helicases from E. coli.     

Biochemical characterization of Escherichia coli DNA helicase I

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single-stranded DNA-stimulated ATPase rate indicates that there is co-operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O-exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low-level reversal of the hydrolytic step during each turnover.     

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.     

The DNA binding properties of the Escherichia coli RecQ helicase

The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.     

Resolving Holliday junctions with Escherichia coli UvrD helicase

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA structures, blunt duplex DNA and RNA-DNA hybrids. Here, we demonstrate that UvrD also catalyzes the robust unwinding of Holliday junction substrates. To characterize this unwinding reaction we have employed steady-state helicase assays, pre-steady-state rapid quench helicase assays, DNaseI footprinting, and electron microscopy. We conclude that UvrD binds initially to the junction compared with binding one of the blunt ends of the four-way junction to initiate unwinding and resolves the synthetic substrate into two double-stranded fork structures. We suggest that UvrD, along with its mismatch repair partners, MutS and MutL, may utilize its ability to unwind Holliday junctions directly in the prevention of homeologous recombination. UvrD may also be involved in the resolution of stalled replication forks by unwinding the Holliday junction intermediate to allow bypass of the blockage.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Study of the ATP-binding site of helicase IV from Escherichia coli

Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding.     

The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels

Summary Recombinant DNA techniques were used to study varibus aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the p _L promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.Abbreviations RBS ribosome binding site - kan kanamycin - Ap ampicillin - Tc tetracycline - CAM chloramphenicol The previous paper in this series is Gilchrist and Denhardt (1987)     

Identification of the gene for DNA helicase II of Escherichia coli

Using a modification of the solid-phase radioimmune assay of Broome and Gilbert [Proc. Natl Acad. Sci. USA, 75, 2746 (1978)] to screen the plaques of lambda recombinant phages for the presence of an elevated level of helicase-II-specific antigen, we have identified the gene for helicase II in a library of Escherichia coli DNA. The DNA selected was subcloned from lambda into plasmid vectors; restriction analysis located the DNA region encoding helicase II in a PvuII fragment identical in size (2900 base pairs) and restriction pattern to that which contains the uvrD gene. Plasmids carrying this DNA fragment complemented the increased sensitivity to ultraviolet irradiation and the mutator phenotype of uvrD mutants. Furthermore, uvrD502 mutant cells were found to liberate no helicase II activity upon extraction. Following transformation with the cloned DNA, active helicase II was recovered from the mutant cells. These results support the view that helicase II is encoded by uvrD.     

The Escherichia coli RecQ helicase functions as a monomer

The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.