INTRODUCTION

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DNA helicases are enzymes involved in a variety of processes such as DNA replication, repair, and recombination, and at least 12 have been identified in Escherichia coli (29, 30). Of these, only DnaB helicase (with 5'-3' polarity) is essential for cell viability, being associated with movement of the chromosome replication fork (27). Three other helicases, Rep, PriA, and UvrD, each with 3'-5' polarity, have also been suggested to participate in chromosome replication, but their roles are not as well established.

The UvrD helicase (also called helicase II) is the product of the uvrD gene and is a polypeptide of 720 amino acids. UvrD is required in the pathways of both methyl-directed mismatch repair (34, 35) and UvrABC-mediated excision repair (46, 47), and uvrD mutants exhibit a spontaneous mutator phenotype as well as increased sensitivity to UV irradiation. Genetic evidence has implicated UvrD in homologous recombination (21, 23, 26, 32). The suggestion has also been made that UvrD participates in DNA replication, based on (i) experiments with cell-free systems (17, 22), (ii) the observation that uvrD null mutations are incompatible with rep (encoding the Rep helicase) or polA (encoding DNA polymerase I) mutations (56), and (iii) identification of dominant-lethal uvrD mutations (8, 59).

In the present study, we discovered that lon (encoding the Lon protease) represents a third locus, mutations in which are incompatible with uvrD null alleles. Chronic induction of the SOS response (reviewed in references 23 and 55) in the uvrD null strains was shown to be the basis for lon incompatibility; other mutations (in dam, priA, mutD, and polA) that exhibited persistent SOS induction were also lon incompatible. We propose a model suggesting a role for the UvrD helicase in removing regions of secondary structure in the lagging strand during replication fork progression, which could account for chronic generation of an SOS-inducing signal in uvrD mutants.

	MATERIALS AND METHODS

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Growth media and conditions. The defined and nutrient media were, respectively, glucose-minimal A and Luria-Bertani (LB) medium (33). cpsB-lac expression was monitored on MacConkey-lactose agar (Difco). Unless otherwise indicated, the growth temperature was 37°C. Isopropyl -D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM. Concentrations of antibiotics and 5-bromo-4-chloro 3-indolyl--D-galactoside (X-Gal) used were as described previously (43, 44).

Bacterial strains and phages. The E. coli strains used are listed in Tables 1 and 2. Phage P1kc was from our laboratory stock. The transposon vehicle phages 1105, 1323, and 1324 for transposition, respectively, of Tn10dKan, Tn10dTet, and Tn10dCm, have been described elsewhere (16). The ordered  phage library of the E. coli genome (19) was obtained from K. Isono.

Plasmids. The plasmid vectors pCL1920 (pSC101 based, encoding Sm^r Sp^r, for general cloning [24]) and pMAK705 (pSC101 based, encoding Cm^r, temperature-sensitive replicon [10]) have been described previously. Plasmids pHYD130 and pHYD141 were constructed in this study and carry the lon⁺ gene subcloned from the  phage 148 of the ordered library of Kohara et al. (19): the former bears a 6-kb insert comprising two contiguous PstI chromosomal segments in the PstI site of vector pCL1920, and the latter bears a 4.1-kb BglII-KpnI chromosomal segment in the BamHI-KpnI sites of vector pMAK705 (see Fig. 1). Plasmids pHYD136 and pHYD138 were also constructed in this study and are described below.

Isolation and characterization of the uvrD400::Tn10dTet mutant. Strain GJ1901, carrying a lacZ(Am) mutation, was subjected to random insertion mutagenesis with the Tn10dTet transposon following infection with 1323, as described elsewhere (16). Tet^r colonies exhibiting increased Lac⁺ papillation frequency were identified on LB agar plates supplemented with 0.1% lactose and X-Gal, as described earlier (43). One such mutant clone was designated GJ1904, and the following characteristics led to the conclusion that it carried a uvrD insertion, designated uvrD400::Tn10dTet. (i) Strain GJ1904 both was UV sensitive and exhibited a high frequency of spontaneous mutations to Lac⁺, nalidixic acid resistance, or rifampin resistance (data not shown). (ii) The Tn10dTet insertion in GJ1904 was linked 30% to the ilv locus in P1 transduction. (iii) Lastly, the uvrD400::Tn10dTet insertion was cloned from the chromosome of GJ1904 as part of a 6.6-kb PstI fragment (size inclusive of the 2.9-kb Tn10dTet element) in the plasmid vector pCL1920, and the resulting plasmid was designated pHYD136. The nucleotide sequence at either junction of Tn10dTet with the E. coli chromosomal DNA was determined by using pHYD136 DNA as a template and the oligonucleotides 5'-TGGTCACCAACGCTTTTCCCGAG-3' and 5'-CTGTTGACAAAGGGAATCATAG-3' as primers (designed so as to read outward from sites immediately internal to the inverted terminal repeat at either end of Tn10dTet); comparison of our results with the E. coli genome sequence (4) permitted the conclusion that the insertion had disrupted the (720-residue) uvrD open reading frame immediately after the second base of codon 476 (data not shown).

Transposon tagging and genetic mapping of uvrD-incompatible mutation in GJ1823. Strain GJ1907 (a spontaneous Lac⁺ revertant of GJ1823) is Kan^r at 30°C but Kan^s at 42°C, because of the presence in GJ1823 (and its retention in GJ1907) of a Kan^r(Ts) insertion near the gal locus (44). GJ1907 was initially transduced to Kan^r at 42°C with a P1 lysate prepared on a population of random Tn10dKan insertion clones of the wild-type strain MG1655. The pool of GJ1907 Kan^r cells was then used as a recipient for transduction to Tet^r with a P1 lysate prepared on the uvrD400::Tn10dTet strain GJ1904. Since the uvrD mutation is ordinarily lethal in GJ1823 and its derivatives (see below), only a small number of Tet^r transductants were recovered in this cross, and these were presumed to represent clones in which the locus conferring incompatibility in GJ1823 had been replaced with the wild-type allele from MG1655 in the first transduction to Kan^r. By this approach, a Kan^r insertion (designated zba-901::Tn10dKan) 83% linked to the locus governing uvrD incompatibility was identified, and it was mapped to the 10-min region (data not shown). Subsequently, the mutation conferring incompatibility with uvrD was itself shown to be 92% cotransducible with a hupB::Kan insertion (data not shown).

Transposon-generated mutation that suppresses uvrD incompatibility in GJ1912. Strain GJ1912 (which carries the uvrD-incompatible mutation) was subjected to random insertion mutagenesis with Tn10dCm following infection with 1324, as described previously (16). The introduction of an uvrD400::Tn10dTet insertion into the pooled culture of Cm^r clones, by P1 transduction, was then attempted. One viable Tet^r transductant colony (GJ1916) so recovered was shown to have a Cm^r insertion that (i) was located in the same 10-min chromosomal region as the locus conferring uvrD incompatibility in GJ1912 (and GJ1823) and (ii) was 100% cotransducible with the phenotype of suppression of uvrD incompatibility. The chromosomal region bearing the Tn10dCm insertion was cloned from GJ1916 on a 3.2-kb PstI fragment in the plasmid vector pCL1920, and the resultant plasmid was designated pHYD138. The oligonucleotide primer 5'-ATTCTGCCTCCCAGAGCCTGA-3', designed to read outward from a site immediately internal to the inverted terminal repeat at one end of Tn10dCm, was used to determine the sequence at the junction of the Tn10dCm insertion in the insert DNA of pHYD138.

Tests for lon incompatibility. Incompatibility of different mutations with lon was established by transduction experiments, wherein it was demonstrated that no colonies were recovered when an attempt was made to introduce an insertion mutation in the concerned locus into a lon strain, or vice versa, unless the recipient carried a plasmid such as pHYD130 bearing the lon⁺ gene. The demonstration in this study (see below) that the lon incompatibility of different mutations could be partially suppressed by growth at 42°C also allowed us to recover transductants from various crosses at the elevated temperature and then test them for a lethality phenotype at 37 or 30°C.

DNA methods. The standard protocols of Sambrook et al. (45) were followed for experiments involving recombinant DNA. Nucleotide sequence determinations were done with the aid of an automated DNA sequencer, using double-stranded plasmid DNAs as templates. The BLAST programs were employed for comparison of DNA sequences with the GenBank sequence database.

Other techniques. Procedures for conjugation (33), P1kc transduction (9), measurement of spontaneous mutation frequency to nalidixic acid or rifampin resistance (43), assay of cell survival following UV radiation (33), and measurement of -galactosidase activity in cultures (33) were as described previously.

	RESULTS

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Identification of a cryptic uvrD-incompatible mutation in strain GJ1823 as lon. The starting point of this study was our observation that a Tn10dTet insertion in codon 476 of the uvrD gene (uvrD400), obtained as described above, could not be transduced into GJ1823, a strain that had been derived (44) by transduction from wild-type MG1655 and that was not expected to carry a uvrD-incompatible mutation. We also confirmed that GJ1823 had not acquired a rep or polA mutation (data not shown); these are the two loci known earlier to be uvrD incompatible.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Molecular characterization of lon-103 and lon-104 mutations. (a) The line at the bottom depicts the physical map of the wild-type lon locus (4, 19) for the enzymes BglII (B), EcoRI (E), KpnI (K), and PstI (P); the open circle and arrow beneath the line denote, respectively, the promoter and transcribed region of the lon gene. Horizontal line segments of the lower and upper inverted triangles represent, respectively, IS186 and Tn10dCm; the vertices denote sites of insertion of these two elements in the lon-103 mutant and the lon-104 suppressor-bearing strain, respectively. The figure is drawn to the scale marked; the distance between the PstI site at the left and the BglII site is 2.42 kb. The contiguous region of DNA (delimited by PstI sites) corresponding to the insert cloned in plasmid pHYD138 is shown by heavy line segments. The recognition site for the Tn10dCm-specific sequencing primer (see the text) is marked by the solid circle. (b) Nucleotide sequence determined with a Tn10dCm-specific sequencing primer using pHYD138 DNA as a template. The extents of Tn10dCm, IS186, and lon sequences are indicated by double-headed arrows above the sequence; dashed lines have been used where the delimited ends do not represent the natural ends of the sequence in question. The right end of IS186 is as specified in the work of Sengstag et al. (50), and the sequence corresponding to the 23-bp right inverted repeat is boxed. The 10 region of the lon promoter (4, 11) is underlined, and the base representing the transcription start site is italicized.

SOS induction in uvrD400::Tn10dTet as basis for lon incompatibility. Another substrate of the Lon protease is the cell septation inhibitor SulA, whose synthesis is induced during the SOS response; as a consequence, lon mutants are known to be sensitive to SOS-inducing agents (reviewed in references 23 and 55). At least some uvrD mutations have been shown earlier to induce the SOS response (3, 25, 38), and we therefore tested the hypothesis that the uvrD-lon combination is rendered inviable because Lon becomes essential to degrade the increased concentration of SulA in the uvrD mutant background. (i) Using sulA-lac expression as a measure of SOS induction, we found that the uvrD400::Tn10dTet insertion (which is lon incompatible) caused a moderate (5-fold) induction of the SOS regulon, as against the 70-fold increase observed in a fully derepressed (lexA) strain (Table 2). The expression value for a merodiploid strain bearing uvrD⁺ on the chromosome and uvrD400 on plasmid pHYD136 was the same as that for haploid uvrD⁺ (Table 2; compare the first and second rows), indicating that the mutation is recessive to the wild-type gene. (ii) Contrarily, the uvrD3 mutation, which does not induce the SOS response (38) (see Table 2), was lon compatible (strain GJ2126). (iii) Finally, the uvrD400::Tn10dTet incompatibility with lon could be suppressed by sulA and lexA3 (Ind) mutations in strains GJ1988 and GJ1989, respectively, in which the inhibitory accumulation of SulA is expected to be abolished (21, 23, 55); predictably, the uvrD400-mediated derepression of sulA-lac was not observed in the lexA3 background (Table 2, GJ1991).

SOS induction is associated with null mutations in uvrD. In order to address the question of whether SOS induction in uvrD strains is the null phenotype, we tested two deletion-insertion uvrD mutations, constructed by Kushner and coworkers (56) and recessive to uvrD⁺, for their effects on reporter gene expression from SOS-responsive promoters and for lon incompatibility. The uvrD288::Kan allele (in which more than two-thirds of the gene has been deleted from its 3' end) was associated with sulA-lac derepression (Table 2). Furthermore, both uvrD288 and the uvrD290::Tet mutation (which represents a deletion of the uvrD promoter and the 5' region of the gene) were lon-103::IS186 incompatible (data not shown). We therefore conclude that SOS induction by uvrD is the null phenotype.

dam, mutD, polA, and priA mutations are also lon incompatible. Mutations in dam, dnaQ (mutD), polA, and priA have previously been shown (or suggested) to lead to chronic SOS induction (1, 36, 39, 40, 48, 53), and we examined whether they are also lon incompatible. We initially confirmed that each of the mutations did lead to a 3- to 40-fold derepression of sulA-lac (Table 2). We then showed, by the criteria listed above, that all four mutations were lon incompatible (dam, dnaQ, and priA were tested with lon-103::IS186; polA was tested with lon-146::Tn10dTet) (data not shown). Suppression of lon incompatibility by sulA was demonstrated for dam and dnaQ (strains GJ1997 and GJ1998, respectively), and suppression of lon incompatibility by lexA3 was demonstrated for priA (strain GJ1999).

lon incompatibility of SOS-inducing mutations is partially suppressed at 42°C. We noticed that the various SOS-inducing mutations such as uvrD400::Tn10dTet, uvrD288, dam, priA, and mutD could be introduced by transduction into a lon strain to give small colonies on plates incubated at 42°C and that the transductants could grow, albeit poorly, upon restreaking at 42°C but not at 30 or 37°C. The degree of sulA-lac derepression by uvrD400::Tn10dTet was unaffected at 42°C (Table 2). On the other hand, cpsB-lac expression in the lon mutant was less pronounced at 42°C (data not shown). Our results support the findings by other workers (13, 15, 51, 57) that alternative proteases such as HslVU (ClpYQ) whose synthesis is induced upon heat shock are able to substitute for Lon in degrading the substrates RcsA and SulA at the elevated temperature.

Dissociation of the uvrD role in SOS induction from its functions in mismatch repair, excision repair, and recombination. The following experiments indicated that SOS induction in uvrD strains is not the consequence of the absence of mismatch repair or nucleotide excision repair. Mutations in mutH or mutS that interfere with mismatch repair did not induce the SOS response, nor did a mutation in uvrC that renders defective the pathway of nucleotide excision repair (Table 2); similar results with mutH, mutL, mutS, and uvrA have been reported earlier (28, 38). A mutH uvrC double mutant in which both pathways are simultaneously nonfunctional (as is the case in uvrD) also was not derepressed for sulA-lac expression (Table 2).

uvrD derivatives of BL21 are very sick. It is known that E. coli B strains are naturally Lon protease deficient. Consistent with the results described above was our observation that uvrD400::Tn10dTet transductants of the E. coli B strain derivative BL21 grow at 37°C as tiny colonies and that they are somewhat more healthy at 42°C. Other workers (8) have earlier reported the construction of UvrD-deficient derivatives of BL21(DE3), but it is possible that these derivatives may inadvertently also have been selected for suppressors of uvrD-lon incompatibility such as sulA.

	DISCUSSION

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We have identified in this study a new set of incompatible pairs of mutations involving the lon gene on the one hand and uvrD, priA, mutD, dam, or polA on the other. The common features that characterize this set are as follows: (i) each of the latter partners of the set results in moderate SOS induction, and (ii) the incompatibilities are suppressed by sulA, lexA3 (Ind), or recA mutations and are partially suppressed by growth at 42°C. Included in the category of lon-incompatible SOS-inducing mutations are both those (such as uvrD, priA, and mutD) for which SOS induction itself is not essential for viability and others (such as dam and polA) for which it is. The simplest interpretation to explain the incompatibility is that persistent SOS induction causes a moderate elevation in levels of SulA protein and that the Lon protease becomes essential for inactivating SulA under these conditions.

The demonstration of lon incompatibility for mutations such as uvrD and priA permits the inference that the latter mutations lead to moderate SOS induction in all (or a majority of) cells in the culture rather than to extreme SOS induction in a smaller subpopulation. The rep mutation appears to be associated with an even lower level of SOS induction than uvrD or priA, since a lon rep mutant is sick yet viable. Chronic SOS induction has also been reported in wild-type cells in the stationary-growth phase (54) but is probably mild enough not to be lethal in a lon mutant. Likewise, mutations in recF (40), ruv (40), or recBC sbcBC (14, 28, 39) have been reported to cause SOS induction, but we found each of them to be lon compatible (data not shown).

It may be noted that the IS186 insertion mutation in the promoter region of lon in strain GJ1823 had arisen spontaneously, in the absence of any imposition of ostensible selection. Ignatov and Chistyakova (11) have also independently reported the identification of a spontaneous IS186 insertion at the same site in the lon promoter. Combined with the fact that wild-type E. coli B isolates are lon negative, our observations raise the question whether natural selection may operate for lon-defective strains under certain conditions.

SOS induction in uvrD mutants probably occurs through a defect in DNA replication. It has been established from earlier studies that the signal for SOS induction in vivo is single-stranded DNA (reviewed in references 23 and 55). Our results indicate that chronic SOS induction in uvrD mutants is the null phenotype. Nevertheless, identification of the precise mechanism is still complicated by the fact that UvrD is involved in several discrete functions and pathways, loss of any one of which could plausibly be modeled to account for SOS induction.

Role for UvrD in lagging-strand replication? In order to account for generation of a moderate SOS-inducing signal in all cells of a culture of a uvrD strain, we propose that UvrD functions as a 3'-5' helicase during lagging-strand replication. The chromosomal replication fork is expected to contain a loop of unreplicated lagging strand that is approximately 1 kb long (27). We suggest that the 3'-5' helicase activity of UvrD helps maintain this single-stranded DNA region free of secondary structure and that in the absence of UvrD, synthesis of some fraction of Okazaki fragments by DNA polymerase III is impeded so that a chronic, low-level SOS-inducing signal is generated by the unreplicated DNA segments on the lagging strand. A similar function was in fact postulated earlier for PriA (29); however, more recent findings have established that the preeminent role of this protein is in the reassembly of collapsed replication forks (7, 18, 58) and that loss of its helicase activity is not sufficient for generation of an SOS-inducing signal in vivo (18, 48).