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.     

Purification of Escherichia coli DNA helicase I from plasmid-transformed cells

DNA helicase I was purified in large quantity from Escherichia coli cells harboring a plasmid that carries the gene encoding helicase I--the traI gene of the F sex factor--cloned in a high copy number vector. Electron microscopic studies on the purified material reveal new properties of the enzyme protein.     

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.     

Concomitant reconstitution of TraI-catalyzed DNA transesterase and DNA helicase activity in vitro

TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

Escherichia coli MutL loads DNA helicase II onto DNA

Previous studies have shown that MutL physically interacts with UvrD (DNA helicase II) (Hall, M. C., Jordan, J. R., and Matson, S. W. (1998) EMBO J. 17, 1535-1541) and dramatically stimulates the unwinding reaction catalyzed by UvrD in the presence and absence of the other protein components of the methyl-directed mismatch repair pathway (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem. 273, 9197-9201). The mechanism of this stimulation was investigated using DNA binding assays, single-turnover helicase assays, and unwinding assays involving long duplex DNA substrates. The results indicate that MutL binds DNA and loads UvrD onto the DNA substrate. The interaction between MutL and DNA and that between MutL and UvrD are both important for stimulation of UvrD-catalyzed unwinding. MutL does not clamp UvrD onto the substrate; and therefore, the processivity of unwinding is not increased in the presence of MutL. The implications of these results are discussed, and models are presented for the mechanism of MutL stimulation as well as for the role of MutL as a master coordinator in the methyl-directed mismatch repair pathway.     

Structure of the DNA repair helicase hel308 reveals DNA binding and autoinhibitory domains

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.     

Image analysis reveals that Escherichia coli RecA protein consists of two domains.

The Escherichia coli RecA protein catalyzes homologous genetic recombination by forming helical polymers around DNA molecules. These polymers have an ATPase activity, which is essential for the movement of strands between two DNA molecules. One obstacle to structural studies of the RecA filament has been that the ATPase results in a dynamical polymer containing a mixture of states with respect to the bound ATP and its hydrolytic products. We have formed filaments which are trapped in the ADP-Pi state by substituting AIF4- for the Pi, and have used these stable filaments to generate a three-dimensional reconstruction from electron micrographs. The resolution of the reconstruction is sufficient to resolve the 38-k RecA subunit into two nearly equal domains. This reconstruction provides the most detailed view yet of the RecA protein, and serves as a framework within which existing biochemical data on RecA can be understood.     

Escherichia coli DNA helicase I. Characterization of the protein and of its DNA-binding properties

Gene traI of the Escherichia coli F sex factor which encodes DNA helicase I was subcloned in a lambda pL-based plasmid vector and expressed in a background of pL non-repressing cells. Neither the non-repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single-stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth-contoured filament with a diameter of 12.5 +/- 0.4 nm and an axial periodicity of 7.0 +/- 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 +/- 0.5 nm and 7.2 +/- 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180,000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates. For comparison, a truncated traI, lacking coding for the amino-terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand-separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase-I-specific sequence of this polypeptide, traI is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon.     

Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.     

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.     

Structure-function analysis of the three domains of RuvB DNA motor protein

RuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Structure-function relationships of domains of the delta subunit in Escherichia coli adenosine triphosphatase.

The topology of the and subunit of the Escherichia coli adenosinetriphosphatase (ECF1) has been explored by proteinase digestion and chemical labeling methods. The delta subunit of ECF1 could be cleaved selectively by reaction of the enzyme complex with very low amounts of trypsin (1:5000, w/w). Cleavage of the delta subunit occurred serially from the C-terminus. The N-terminal fragments of the delta subunit remained bound to the core ECF1 complex through sucrose gradient centrifugation, indicating that part of the binding of this subunit involves the N-terminal segment. ECF1, in which around 20 amino acids had been removed from the C-terminus of delta, still bound to ECF0 but DCCD sensitivity of the ATPase activity was lost. When ECF1 was reacted with N-ethyl[14C]maleimide ([14C]NEM) in the native state, only one of the two Cys residues on the delta subunit was modified. This residue, Cys-140, was also labeled in ECF1F0. Cys-140 was shown to be involved in the disulfide bridge between alpha and delta subunits that is generated when ECF1 is treated with CuCl2. Thus, the C-terminal part of the delta subunit around Cys-140 can interact with the core ECF1 complex. These results suggest a model for the delta subunit in which the central part of polypeptide is a part of the stalk, with both N- and C-termini associated with ECF1.     

Escherichia coli DNA helicase II is active as a monomer

Helicases are thought to function as oligomers (generally dimers or hexamers). Here we demonstrate that although Escherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (Kd = 3.4 microM), the protein is active in vivo and in vitro as a monomer. A mutant lacking the C-terminal 40 amino acids (UvrDDelta40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrDDelta40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.     

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.     

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.     

Escherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zinc-binding domains

Escherichia coli DNA topoisomerase I catalyzes interconversions of different DNA topological isomers by the breakage and rejoining of DNA phosphodiester bonds. It has a crucial role in maintaining an optimal DNA superhelicity in E. coli. It is a single polypeptide of 864 amino acids. Analysis of the amino acid sequence reveals three tandem repeat units each containing two pairs of cysteines suggesting that each unit may form a zinc-binding domain. We have determined that each enzyme molecule contains three to four zinc atoms using inductively coupled plasma-atomic emission analysis. Modification of the cysteine residues and removal of the zinc from the enzyme result in loss of activity. Zinc ions are needed for full recovery of enzyme activity when the cysteine modification is reversed. Comparison with the zinc-binding domains of the sequence-specific DNA-binding proteins shows significant differences.