Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

Characterization of DNA helicase II from a uvrD252 mutant of Escherichia coli.

The loss of DNA helicase II (UvrD) in Escherichia coli results in sensitivity to UV light and increased levels of spontaneous mutagenesis. While the effects of various uvrD alleles have been analyzed in vivo, the proteins produced by these alleles have not been examined in any detail. We have cloned one of these alleles, uvrD252, and determined the site of the mutation conferring the phenotype. In addition, the protein it encodes has been purified to homogeneity and characterized in vitro. The mutation responsible for the phenotype was identified as a glycine-to-aspartic-acid change in the putative ATP-binding domain. In comparison to wild-type DNA helicase II, the UvrD252 enzyme exhibited reduced levels of ATPase activity and a large increase in the Km for ATP. The ability of UvrD252 to unwind DNA containing single-stranded regions, as well as DNA containing only nicks, was reduced in comparison to that of the wild-type enzyme. Possible interpretations of these results in relation to the phenotypes of the uvrD252 mutant are discussed. This represents the first detailed analysis of the biochemical properties of a mutant DNA helicase II protein.     

Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction.

Site-directed mutagenesis has been employed to address the functional significance of the highly conserved aspartic and glutamic acid residues present in the Walker B (also called motif II) sequence in Escherichia coli DNA helicase II. Two mutant proteins, UvrDE221Q and UvrDD220NE221Q, were expressed and purified to apparent homogeneity. Biochemical characterization of the DNA-dependent ATPase activity of each mutant protein demonstrated a kcat that was < 0.5% of that of the wild-type protein, with no significant change in the apparent Km for ATP. The E221Q mutant protein exhibited no detectable unwinding of either partial duplex or blunt duplex DNA substrates. The D220NE221Q mutant, however, catalyzed unwinding of both partial duplex and blunt duplex substrates, but at a greatly reduced rate compared with that of the wild-type enzyme. Both mutants were able to bind DNA. Thus, the motif II mutants E221Q and D220NE221Q were able to bind ATP and DNA to the same extent as wild-type helicase II but demonstrate a significant reduction in ATP hydrolysis and helicase functions. The mutant uvrD alleles were also characterized by examining their abilities to complement the mutator and UV light-sensitive phenotypes of a uvrD deletion mutant. Neither the uvrDE221Q nor the uvrDD220NE221Q allele, supplied on a plasmid, was able to complement either phenotype. Further genetic characterization of the mutant uvrD alleles demonstrated that uvrDE221Q confers a dominant negative growth phenotype; the uvrDD220NE221Q allele does not exhibit this effect. The observed difference in effect on viability may reflect the gene products' dissimilar kinetics for unwinding duplex DNA substrates in vitro.     

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 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.     

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.     

Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination.

A yeast strain was constructed that had a disruption of the chromosomal RAD3 gene and carried a series of centromeric plasmids with defined mutations in this gene. Using this isogenic collection, we examined sensitivity to UV radiation, spontaneous and UV radiation-induced mutagenesis, and mitotic recombination. Several alleles resulted in a marked increase in UV sensitivity. Most of these alleles were found to carry mutations located in consensus motifs for DNA helicases. Other alleles caused a modest or no increase in UV sensitivity and carried mutations in regions of the Rad3 polypeptide that are apparently not conserved. This correlation suggests that the DNA helicase activity of Rad3 protein is required for nucleotide excision repair of DNA. Some rad3 alleles conferred a marked increase in the frequency of spontaneous mutagenesis, including nonsuppressor reversion of the lys2-1 ochre mutation. These alleles also showed a good correlation with conserved DNA helicase domains, suggesting that the Rad3 DNA helicase also plays a role in the fidelity of DNA synthesis or postreplicative mismatch correction. Several rad3 mutator alleles also resulted in increased levels of mitotic recombination. Increased spontaneous mutagenesis and mitotic recombination are characteristic features of the Rem- phenotype. However, in contrast to the prototypic Rem- phenotype, the rad3 mutator alleles identified in this study did not confer inviability in the presence of mutations in the RAD50 or RAD52 gene required for strand break repair of DNA.     

DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.     

Identification and Characterization of Escherichia coli DNA Helicase II Mutants That Exhibit Increased Unwinding Efficiency

Using a combination of both ethyl methanesulfonate and site-directed mutagenesis, we have identified a region in DNA helicase II (UvrD) from Escherichia coli that is required for biological function but lies outside of any of the seven conserved motifs (T. C. Hodgman, Nature 333:22–23, 1988) associated with the superfamily of proteins of which it is a member. Located between amino acids 403 and 409, alterations in the amino acid sequence DDAAFER lead to both temperature-sensitive and dominant uvrD mutations. The uvrD300 (A406T) and uvrD301 (A406V) alleles produce UV sensitivity at 44°C but do not affect sensitivity to methyl methanesulfonate (MMS). In contrast, the uvrD303 mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is dominant to uvrD⁺ when present at six to eight copies per cell. Several of the alleles demonstrated a strong antimutator phenotype. In addition, conjugal recombination is reduced 10-fold in uvrD303 strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (uvrD302) was neutral. To determine the biochemical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a uvrDΔ294 deletion background and characterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild-type control, while its K_m for ATP binding remains unchanged. More importantly, the UvrD303 protein unwinds partial duplex DNA up to 10 times more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propose that the region located between amino acids 403 and 409 serves to regulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA repair systems in which the protein participates.     

Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517(am) as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517(am) derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor ``srjD' is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.     

ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.     

A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) is essential for DNA repair and recombination

Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) by site-directed mutagenesis. These mutations affected the helicase activity and the ATP-dependent exonuclease activity on double-stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single-stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid-soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.     

Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli.

The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase II) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance. Each deletion was verified by Southern blots, and the location of each deletion was confirmed by P1-mediated transduction. Cell strains containing the single and double deletions were viable, indicating that helicases II and IV are not essential for viability. Cell strains lacking helicase IV (delta helD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain. As expected, cell strains containing the helicase II deletion (delta uvrD) were sensitive to both UV irradiation and MMS. The introduction of the helicase IV deletion into a delta uvrD background had essentially no effect on the UV and MMS sensitivity of the cell strains analyzed. The double deletions, however, conferred a Rec- mutant phenotype for conjugational and transductional recombination in both recBC sbcB(C) and recBC sbcA backgrounds. The Rec- mutant phenotype was more profound in the recBC sbcB(C) background than in the recBC sbcA background. The recombination-deficient phenotype indicates the direct involvement of helicase II and/or helicase IV in the RecF pathway [recBC sbcB(C) background] and RecE pathway (recBC sbcA background) of recombination. The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient. In addition, helicase IV has been overexpressed in a tightly regulated system. The data suggest that even modest overexpression of helicase IV is lethal to the cell.     

A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding

The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDDelta107C (deletion of the last 107 C-terminal amino acids), UvrDDelta102C, and UvrDDelta40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDDelta107C and UvrDDelta102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDDelta40C protein fully complemented the loss of helicase II in both repair pathways. UvrDDelta102C and UvrDDelta40C were purified to apparent homogeneity and characterized biochemically. UvrDDelta102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (kcat = 0.01% of the wild-type level). UvrDDelta40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.     

The cockayne syndrome group B gene product is involved in cellular repair of 8-hydroxyadenine in DNA

Cockayne syndrome (CS) is a human disease characterized by sensitivity to sunlight, severe neurological abnormalities, and accelerated aging. CS has two complementation groups, CS-A and CS-B. The CSB gene encodes the CSB protein with 1493 amino acids. We previously reported that the CSB protein is involved in cellular repair of 8-hydroxyguanine, an abundant lesion in oxidatively damaged DNA and that the putative helicase motif V/VI of the CSB may play a role in this process. The present study investigated the role of the CSB protein in cellular repair of 8-hydroxyadenine (8-OH-Ade), another abundant lesion in oxidatively damaged DNA. Extracts of CS-B-null cells and mutant cells with site-directed mutation in the motif VI of the putative helicase domain incised 8-hydroxyadenine in vitro less efficiently than wild type cells. Furthermore, CS-B-null and motif VI mutant cells accumulated more 8-hydroxyadenine in their genomic DNA than wild type cells after exposure to gamma-radiation at doses of 2 or 5 Gy. These results suggest that the CSB protein contributes to cellular repair of 8-OH-Ade and that the motif VI of the putative helicase domain of CSB is required for this activity.     

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.     

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis , in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus . This gene is located at 55° on the chromosome and belongs to a putative operon together with a ligase gene ( lig ) and two unknown genes named pcrB and yerH . As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli . PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep⁻ phenotype on E. coli . Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.     

Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication

The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R(376)KNGK(380), abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R(184)KR(186) and D(436)GEE(439), we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D(334)EE(336) and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37 degrees C) and showed reduced RNA synthesis at the higher temperature.