Construction and analysis of deletions in the structural gene (uvrD) for DNA helicase II of Escherichia coli.

DNA helicase II, the product of the uvrD gene, has been implicated in DNA repair, replication, and recombination. Because the phenotypes of individual uvrD alleles vary significantly, we constructed deletion-insertion mutations in the uvrD gene to determine the phenotype of cells lacking DNA helicase II. Deletion mutants completely lacking the protein, as well as one which contains a truncated protein retaining the ATP-binding site, remained viable. However, they were sensitive to UV light and exhibited elevated levels of homologous recombination and spontaneous mutagenesis. In addition, mutations mapping in or near rep which allow construction of rep uvrD double mutants at a high frequency were isolated.     

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I (helicase II)

Three mutants producing thermosensitive DNA-dependent Adenosine triphosphatase (ATPase) I were screened from a collection of temperature-sensitive mutants of Escherichia coli K12. ATPase I purified to near homogeneity from one of the mutants (JE11000) possesses both thermosensitive DNA-dependent ATPase and DNA helicase activities. We have shown that ATPase I is encoded by the uvrD gene as first suggested by Oeda et al. (1982): (i) the thermosensitive ATPase I mutation present in JE11040 lies in or very close to the uvrD gene, (ii) ATPase I activity is absent in uvrD210, uvrD156, and uvrD252 mutants. Thus the thermosensitive mutations correspond to new uvrD mutations. However, the mutation present in JE11040 confers neither UV sensitivity nor mutator phenotype at high temperature. Evidence is presented that the mutant ATPase I is stabilized in vivo at 42 degrees C.     

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.     

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.     

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.     

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.     

Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.     

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.     

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.     

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.     

Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12

The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication. A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant. Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant. The strain showed no Weigle reactivation. From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant. It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.     

Direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II

The direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II was studied using gapped linear DNA molecules with short duplex ends as substrate. The results suggest that DNA helicase II unwinds with 3'-5' polarity relative to the single strand of the DNA partial duplex. At high enzyme DNA ratio the enzyme also unwinds the duplex connected to the 3' end of the single strand and, as further studies show, fully duplex linear DNA. The fraction of DNA unwound decreases as the length of the duplex substrate increases. The preference of DNA helicase II for a short duplex can obscure the fact that the typical substrate is duplex connected to the 5' end of a single strand.     

Intragenic suppression in the uvrD gene of Escherichia coli. I. Temperature-sensitive uvrD mutations

A temperature-sensitive uvrD mutant, HD323 uvrD4, was isolated from the uvrD mutant HD4 uvrD3. The temperature sensitivity of the uvrD4 gene product was reversible. The suppressor mutation uvrD44 which rendered the uvrD3 mutant temperature-sensitive could be separated from the uvrD3 mutation by replacing the PstI fragment, which encodes the C-terminal half of the UvrD protein. The uvrD44 mutation was found to make host bacteria lethal at non-permissive temperatures only when cloned on a low copy vector pMF3. The nucleotide sequence of the uvrD3 and uvrD4 mutant genes was determined. The nucleotide change found in the uvrD3 at +1235, GAA to AAA, only alters the amino acid sequence from Glu at 387 to Lys. The uvrD44 has another nucleotide change at +1859, GAA to AAA (Glu at 595 to Lys), which is considered to be the suppressor mutation uvrD44.     

Characterization of a mutant form of ribosomal protein S1 from Escherichia coli.

An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized. The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1. Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA. Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis.