Tandem Repeat Recombination Induced by Replication Fork Defects in Escherichia coli Requires a Novel Factor, RadC

Corresponding author: Susan T. Lovett, Rosenstiel Basic Medical Sciences Center MS029, Brandeis University, Waltham, MA 02454-9110., lovett{at}hydra.rose.brandeis.edu (E-mail)

Communicating editor: R. MAURER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB⁺ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork.

AN essential E. coli gene, dnaB, encodes the hexameric helicase associated with the DNA polymerase III replication fork (reviewed in MARIANS 1996 ). The DnaB protein is connected to most of the enzymatic activities taking place within the Pol III replisome. To facilitate its loading onto DNA, it associates with the DnaC primosomal (WICKNER and HURWITZ 1975 ; KOBORI and KORNBERG 1982 ) and DnaA initiation proteins (MARSZALEK and KAGUNI 1994 ). It interacts with the -subunit of the Pol III holoenzyme to solidify the processivity of the replication complex (DALLMANN et al. 1995 ; KIM et al. 1996 ) and with primase to initiate each Okazaki fragment on the lagging strand of the fork (TOUGU and MARIANS 1996 ). It exhibits 5' to 3' helicase activity (LEBOWITZ and MCMACKEN 1986 ) and DNA-dependent ATPase activity (ARAI and KORNBERG 1981 ). DnaB protein plays multiple roles as helicase, regulator of primer synthesis, and processivity factor. Errors in any of these functions could hinder replication and potentially stall the fork or leave unsynthesized or incorrectly synthesized DNA strands that require repair.

Previously we reported that a dnaB107(ts) mutation, at its permissive temperature, elevates the deletion rate of a tandemly repeated sequence (SAVESON and LOVETT 1997 ). This hyperdeletion phenotype was almost entirely dependent on recA, which indicates a recombinational mechanism. The exact defect in dnaB107 mutants at their permissive temperature is unknown; they synthesize Okazaki fragments at 30° but their overall ability to synthesize DNA is reduced (LARK and WECHSLER 1975 ; SCLAFANI and WECHSLER 1981B ). The defect in this strain may result in an unstable replication fork, prone to stalling or dissociation.

Escherichia coli contains a number of systems used to repair damaged DNA, including several recA-dependent DNA recombination pathways (CLARK and SANDLER 1994 ). E. coli recombination (rec) mutants typically are sensitive to UV irradiation, presumably because these enzymes are needed to repair blocked replication forks. Strains undergoing replication are more UV-sensitive than nonreplicating strains because DNA synthesis is blocked by UV lesions, which leaves regions of single-strand DNA (ssDNA) and stalled forks prone to breakage (RUPP 1996 ). Even without exogenous DNA damage, difficulties during normal DNA replication may also lead to ssDNA gaps and stalled replication forks. DNA recombination enzymes are used to repair both ssDNA gaps and double-strand breaks (DSBs), and the mechanisms of these two types of repair are genetically distinguishable. DSB repair requires recB, recC, and recD, whereas the majority of single-strand gap repair uses the recF, recO, and recR genes (SMITH et al. 1987 ).

Arrested replication forks have been shown to stimulate recombination. They provide nascent DNA ends and ssDNA regions, both of which can be substrates for recombination (BIERNE and MICHEL 1994 ). Stalled replication forks can cause accumulation of linear DNA (MICHEL et al. 1997 ), indicative of fork breakage, which may be caused either by an endonuclease, by replication of incomplete daughter-strands (SKALKA 1974 ), or by physical breakage associated with premature chromosome segregation (KUZMINOV 1995 ). The terminus region of the E. coli chromosome is a hotspot for recA-dependent deletion events (LOUARN et al. 1991 ), where replication fork progression is arrested at ter sites by the antihelicase activity of bound Tus protein (KHATRI et al. 1989 ; LEE et al. 1989 ). Blocked replication can promote RecA, RecBCD-dependent recombination when the ter site is moved elsewhere on the E. coli chromosome (HORIUCHI and FUJIMURA 1995 ).

In this article we investigate the mechanism of chromosomal deletion events stimulated in dnaB107 mutants at their permissive temperature of growth. This stimulation was previously found to be almost entirely dependent on recA, the master recombination gene of E. coli, which indicates a recombinational mechanism for deletion formation (SAVESON and LOVETT 1997 ). Many genes affecting conjugational recombination have been isolated in E. coli (CLARK and SANDLER 1994 ) but in most cases have not been tested for effects on recombination stimulated by replication fork defects. The deletion formation stimulated by dnaB107 gives us a unique opportunity to examine this type of recombination specifically. We report here that dnaB107 hyperdeletion does not require recF, recO, and recR genes known to be involved in recombinational gap repair. Instead, we found deletions depended partially on the DSB repair gene recB and almost entirely on the radC gene. The radC gene has not been extensively characterized: its biochemical function is unknown but it mediates a growth-medium-dependent component of DNA repair following X- and UV-irradiation (FELZENSZWALB et al. 1984 , FELZENSZWALB et al. 1986 ). The inhibition of dnaB107-induced deletion events constitutes a new phenotype for radC and suggests that RadC plays an important role in DNA recombination of blocked or damaged replication forks.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains, plasmids, and growth:
Strains were grown as previously described on Luria broth (LB) medium: 1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodium chloride, 0.0005% thymine, and, for plates, 1.5% agar (WILLETTS et al. 1969 ). All strains were grown at 30° except for the dnaB ruvC and dnaB recB mutant strains, which were constructed and grown at 25°. For transductions and preparation of P1 phage lysates, cultures were grown in LCG media: LB media supplemented with 1% glucose and 2 mM calcium chloride and, for plates, 1% agar. LCG top agar contained 0.7% agar. The following antibiotic concentrations were used: ampicillin (Ap) at 100 µg/ml, tetracycline (Tc) at 7–15 µg/ml, kanamycin (Km) at 30–60 µg/ml, and chloramphenicol (Cm) at 15 µg/ml. Isogenic strains in AB1157 background were constructed by P1 transduction (MILLER 1992 ) at 30° or 25° and are listed in Table 1. To determine the extent of temperature-dependent lethality, various dnaB derivatives were grown on LB + Ap plates at 25°. Whole colonies were picked and grown in LB + Ap broth for 4 hr. Cultures were serially diluted in 56/2 buffer (WILLETTS et al. 1969 ) and plated on LB + Ap medium. Plates were incubated at various temperatures, and percentage survival was determined by dividing the number of cfu at the appropriate temperature by that at 25°.

To construct heterozygous strains for dnaB, the wild-type Salmonella dnaB gene was cloned into plasmid vector pBCSK (Stratagene, La Jolla, CA) to produce plasmid pSTL265. This plasmid encodes Cm^r and is derived from pUC19 with a copy number of 500–700 per cell. To create pSTL265, the 2.0-kb HindIII EcoRI fragment of pFF17 (WONG et al. 1988 ) was ligated with pBCSK and transformed by electroporation into XL1-Blue (F'::Tn10 proAB lacIq lacZM15/recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac; Stratagene) that was made electrocompetent (DOWER et al. 1988 ). Cm^r LacZ^- colonies were selected on LB + Cm + X-gal (50 µg/ml) + 1 mM IPTG medium. Plasmid DNA was purified by alkaline lysis (SAMBROOK et al. 1989 ) and was transformed by electroporation into the appropriate strains by selection for Cm resistance and screened for the ability to complement dnaB107 for growth at 42°. Strains carrying pSTL265 were grown in the presence of Cm for viability and deletion assays.

Chromosomal deletion assays:
Deletions were selected using an insertion of bla and tetAdup787 in the lacZ gene on the chromosome as previously described (LOVETT et al. 1993 ). The tetAdup787 allele contains an internal repeated sequence of 787 bp, from the EcoRV site to the NruI site of tetA, which inactivates the gene. A precise deletion of one tandem repeat results in a functional tetA gene that confers tetracycline-resistance to the cell. Assays were performed as previously described (SAVESON and LOVETT 1997 ). For each mutant strain, single whole colonies were picked from LB + Ap plates and grown in LB broth for 4 hr at 30°. Strains containing pSTL265 were grown on LB + Ap + Cm plates and LB or LB + Cm broth. Cultures were diluted in 56/2 buffer and the number of Ap^r and Tc^r cells in the population was determined by plating on selective medium. Retention of the pSTL265 plasmid was confirmed by also plating dilutions on LB + Cm. Deletion rates were calculated by the method of the median (LEA and COULSON 1949 ), and 95% confidence intervals were determined as previously described (WIERDL et al. 1996 ; SAVESON and LOVETT 1997 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Elevated deletion phenotype is specific to the dnaB107 mutant:
Deletion between tandem repeats in dnaB107 mutants is greatly elevated relative to wild-type strains (SAVESON and LOVETT 1997 ). We have speculated that this elevation could be due either to helicase errors resulting in stalled replication forks or to disruption of the helicase-primase interaction required for lagging-strand replication. We determined deletion rates in two other temperature-sensitive dnaB mutants, dnaB252 and dnaB22, and in a primase, dnaG2903, mutant strain. In contrast to dnaB107, which stimulates deletion formation ~300-fold, these latter three mutations showed little or no effect on deletion rates (Table 2). The elevated dnaB107 deletion phenotype is therefore allele-specific and may reflect a specific defect that results in stalled or broken replication forks that stimulate deletion events.

The hyperdeletion phenotype of dnaB107 is semidominant:
The presence of the wild-type Salmonella typhimurium dnaB gene, carried on a high-copy plasmid in our dnaB107 E. coli strains, supported growth at 42° (Table 3). We used this homeologous Sty dnaB⁺ gene instead of the E. coli gene to prevent recombination between plasmid and chromosome (RAYSSIGUIER et al. 1989 ), which would result in loss of the dnaB107 mutation during our experiments. The Salmonella dnaB sequence is 85% identical at the nucleotide level and 93% identical at the amino acid level to the E. coli sequence (WONG et al. 1988 ) and is expressed from its native promoter in this plasmid construct.

In dnaB107/pSty dnaB⁺ heterozygous strains, deletion events at 30° were still 70-fold elevated over that seen in a dnaB⁺/pSty dnaB⁺ control strain (Table 4), although this level is ~10-fold lower than that of the strain carrying dnaB107 alone (Table 2). Thus the hyperdeletion phenotype of dnaB107 is semidominant to the Salmonella dnaB⁺ gene. This semidominant effect was also seen in a dnaB107 strain heterozygous for the E. coli dnaB⁺ gene. Deletion rates were similarly elevated in a dnaB107/pEco dnaB⁺ heterozygous strain, within twofold of the value in the dnaB107/pSty dnaB heterozygote (C. J. SAVESON and S. T. LOVETT, unpublished results). Thus, dnaB107 is an example of a mutation with both recessive and dominant genetic effects. The presence of DnaB⁺ helicase proteins allows for more efficient replication and improved viability, but this dominance indicates that some mutant DnaB107 helicase proteins must load onto DNA (either as a homogenous complex or mixed with DnaB⁺ subunits in the hexamer) and function abnormally to stimulate deletions.

Chromosomal deletion in dnaB107 strains is recFOR-independent but dependent on a minor repair gene, radC:
To determine the mechanism of deletion events in dnaB107 strains, we assayed the genetic dependence of the hyperdeletion phenotype. Deletion rates for various recombination mutant derivatives are shown in Table 5. Mutations in recF, recO, and recR appear to have little effect on the dnaB107 rate, whereas a recN mutation may lower deletion rates a modest twofold. RecF, RecO, and RecR proteins promote and stabilize RecA filament formation on recombination substrates (UMEZU et al. 1993 ; WEBB et al. 1995 ; SHAN et al. 1997 ) and have been implicated in gap-filling repair (SMITH et al. 1987 ) and replication restart (COURCELLE et al. 1997 ).

A mutation in radC all but eliminated the increased deletion rate in dnaB107 (Table 5). The double mutant of radC and dnaB107 showed a 90-fold lowered deletion rate compared to the dnaB107 single mutant and was comparable to that of the recA dnaB107 strain. A radC mutation did not lower but, in fact, slightly elevated deletion rates in dnaB⁺ strains. The biochemical function of RadC is unknown, but is responsible for a minor component of postreplication repair and joining of strand breaks after UV damage (FELZENSZWALB et al. 1984 , FELZENSZWALB et al. 1986 ). Our results suggest that RadC, similar to RecA, plays an essential role in promoting the recombination that accompanies replication fork defects.

Synthetic lethality of dnaB ruvC and dnaB recB:
Although the hyperdeletion phenotype in dnaB107 mutants was insensitive to mutations affecting gap-filling repair, it may be promoted by breakage of the replication fork and subsequent recombinational repair of double-strand breaks. Indeed, broken chromosomal DNA has been detected in dnaB8 mutants at their nonpermissive temperature of growth (MICHEL et al. 1997 ). Such DSB repair requires the recBCD genes of E. coli, which encode an exonuclease/helicase that processes double-strand breaks and facilitates their recombination (TAYLOR 1988 ). In addition to recBCD, we also wished to test the effects of a mutation in the Holliday junction resolvase gene, ruvC, on dnaB107 hyperdeletion. However, we encountered difficulties in constructing the double-mutant strains dnaB107 recB2053 and dnaB107 ruvC53. Poorly growing transductants were obtained at 25° and both double-mutant combinations were shown to be lethal at 30° (Table 3). In contrast, the single dnaB107 mutant grew at temperatures up to 32° (C. J. SAVESON and S. T. LOVETT, unpublished data). Lethality of dnaB8 recB mutants has been previously reported, but only at the dnaB8 nonpermissive temperature (MICHEL et al. 1997 ). The synthetic lethality of dnaB107 with ruvC and recB mutations suggests that as dnaB107 strains approach their nonpermissive temperature, they require increased processing of branched intermediates and double-strand DNA ends to maintain cellular viability.

The effects of ruvC and recB on hyperdeletion in dnaB107/dnaB⁺ heterozygous strains:
Because the viability of dnaB107 ruvC and dnaB107 recB was so poor, we could not determine deletion rates in these strains, even at 25°. However, transformation with pSTL265, containing Salmonella dnaB⁺, relieved the hypertemperature sensitivity of both double-mutant strains, which allowed growth at 30°. With these dnaB⁺/dnaB107 heterozygous strains we were able to perform deletion assays at 30°. Deletion rates were still enhanced in the dnaB107 ruvC/pdnaB⁺ mutant to the same extent as the dnaB107/pSty dnaB⁺ strain (Table 4). Deletion rates in the dnaB107 recB/pSty dnaB⁺/strain were reduced fivefold.

These results suggest that the recA-dependent deletion events stimulated by dnaB107 are only partially dependent on Exonuclease V (RecBCD), and they do not require the Holliday junction resolving activity of RuvC endonuclease. However, the inviability of the dnaB107 recB and dnaB107 ruvC strains suggests that, indeed, ExoV and RuvC play an important role in dnaB107 mutant survival. We conclude that dnaB107 may be creating an environment where stalled forks are broken and must be repaired to maintain the health of the strain, but in all likelihood RecBCD-mediated recombination is not the only pathway for elevated deletions in this mutant.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have shown that a mutation within DnaB, the replication fork helicase of E. coli, stimulates RecA-dependent recombination between tandem repeats in a semidominant and allele-specific manner. Aberrant replication in the presence of the mutant DnaB107 fork helicase may therefore initiate recombinational repair events that occasionally culminate in rearrangements between tandemly repeated sequences. This hyperrecombination phenotype conferred by dnaB107 provided us with a unique opportunity for genetic analysis to elucidate the mechanism of recombination within replication forks.

The most novel result of our analysis was the identification of the radC gene as essential for dnaB107-stimulated recombination between tandem repeats. The specific function of RadC in recombination and repair is unknown. There is only one allele of radC, which has been isolated as a mutation enhancing X-irradiation sensitivity (FELZENSZWALB et al. 1984 ). However, radC is not a general repair gene (as are recA, recB, recC, and recN); its effect requires certain conditions of growth. Cells growing in rich medium are more resistant to X-irradiation than cells growing in minimal medium; this component of repair ["medium-dependent repair" (MDR)] requires radC (FELZENSZWALB et al. 1984 ). A mutant in radC is also sensitive to UV-irradiation, and radC is epistatic to recA, suggesting a role in some aspect of recombinational DNA repair (FELZENSZWALB et al. 1984 ). As assayed by sucrose gradient centrifugation of postirradiation DNA, the radC mutant shows partial defects in single-strand break and DSB repair (FELZENSZWALB et al. 1986 ). Although our results showed that radC was required for recombination between tandem repeats stimulated by dnaB107, it was not required for tandem repeat recombination in dnaB⁺ strains, nor was it previously found to be required for recombination after conjugation (FELZENSZWALB et al. 1984 ). Rates of tandem repeat recombination in dnaB107 and dnaB⁺ strains were not affected by minimal medium growth (C. J. SAVESON and S. T. LOVETT, unpublished results), which indicates that the radC-dependent recombination pathway is not simply induced by rich medium growth. Rather, because cells growing in rich medium contain multiple replication forks due to reinitiation of replication before completion of the previous cycle (VON MEYENBURG and HANSEN 1987 ), the requirement of radC specifically for MDR, as well as our observations concerning radC, are consistent with the idea that RadC's action is restricted to recombination between sister chromosomes or replication forks. Similar recA-dependent DNA recombination processes may be required when a cell repairs radiation damage in the fork or blocked replication caused by the dnaB107 mutation. Further analysis of RadC should reveal what role it plays in recombinational repair.

Opportunities for tandem repeat deletion by misalignment of substrates can arise during recombinational repair of blocked forks either via a gap-filling (Figure 1) or DSB repair (Figure 2) mechanism. Our genetic analysis suggests that dnaB107-stimulated deletion formation occurs, at least in part, by DSB-mediated events. RecFOR proteins are required for gap-filling repair, but mutations in recF, recO, or recR did not affect hyperdeletion in dnaB107 mutants. The breakage of stalled replication forks in dnaB107 mutants is expected to require RecBCD-mediated repair of the broken DNA ends. Because hyperdeletion in dnaB107/dnaB⁺ heterozygous strains was reduced fivefold by a mutation in recB, DSB break repair may be the major contributor to recombination stimulated by dnaB107. Breakage of the E. coli chromosome to a linear form has been observed in various strains where replication fork progression is inhibited, which include rep and dnaB helicase mutants and a strain with abnormally positioned ter sequences (MICHEL et al. 1997 ). This DSB-mediated recombination may require, in part, RecN, of unknown biochemical function, which has been implicated in DSB repair (PICKSLEY et al. 1984 ; SARGENTINI and SMITH 1986 ). However, even the dnaB107/dnaB⁺ recB mutant showed a 10-fold higher deletion rate than wild-type strains, which indicates that recB-independent recombination is also stimulated by dnaB107. This may be due to processing of DSBs by nucleases other than RecBCD or may reflect gap-filling recombination mediated by RecFOR. Because of the lethality of the dnaB107 recB double mutant, we were unable to construct a recB recF dnaB107 triple mutant to test this latter hypothesis. It is also possible that a substantial amount of deletion in dnaB107 strains occurs independently of both the RecBCD and the RecFOR pathways of recombinational repair via an unknown mechanism requiring RecA, RadC, and potentially other functions.

View larger version (18K): In this window In a new window Download PPT slide   Figure 1. Tandem repeat deletion by a gap-repair mechanism. (A) Incomplete replication of a chromosomal segment carrying a tandem repeat, in black, (B) initiates a recombinational gap-filling reaction. (C) Nicking and branch-migration of the crossed strands, followed by replication into the gap, produces an unequal crossing-over intermediate, which can be resolved to (D) recombinant products that carry either a deletion or a triplication of the repeat.

View larger version (18K): In this window In a new window Download PPT slide   Figure 2. Tandem repeat deletion by a double-strand break repair mechanism. (A) Incomplete replication of a chromosomal segment carrying a tandem repeat, in black, (B) can lead to chromosomal breakage. (C) The broken fork can be repaired by recombination with the sister chromosome. (D) If invasion occurs at the repeat, the fork may be reestablished with a deletion formed on one sister chromosome.

We found that dnaB107 was synthetically lethal with recB or ruvC, but not with recA, recF, or radC. The increased temperature sensitivity of the double mutants recB dnaB and ruvC dnaB suggests that, as dnaB107 strains approach their nonpermissive temperature, they require increased processing of branched intermediates and double-strand DNA ends to maintain viability. It must be the accumulation of these intermediates that causes lethality rather than a failure to recombine, as mutations in recA and radC, which completely block dnaB107-stimulated recombination, are quite viable in combination with dnaB107. Unrepaired gaps may be broken and then become substrates for RecBCD-mediated degradation or recombination; recB mutants may accumulate broken forks that the cell may be unable either to degrade or to recombine. Double dnaB107 recA or dnaB107 radC mutants may be unable to recombine but may be still able to degrade broken chromosomes via RecBCD. Thus DNA degradation via RecBCD may be crucial for viability of dnaB107 mutants, but RecABCD-mediated recombination is not (Figure 3). [A similar hypothesis has been offered to explain the fact that rep helicase mutants are inviable with recB but not with recA (MICHEL et al. 1997 ).] ruvC mutants may become trapped in intermediates they cannot resolve. These intermediates may stall advancing replication forks in a lethal manner.

View larger version (13K): In this window In a new window Download PPT slide   Figure 3. Alternative processing of broken forks by RecBCD. (A) A broken sister chromosome in the replication fork may be eliminated by (B) RecBCD degradation. (C) Alternatively, the fork can be repaired by recombination aided by RecBCD, RecA, and RadC proteins. In both cases, the remaining gaps would be filled by repair replication and ligation. Mutations in recBCD block both pathways and may therefore cause synthetic lethality with dnaBts; mutations in recA and radC block only the rightward pathway and the cell may escape killing by RecBCD-mediated degradation.

Although dnaB107, dnaB22, and dnaB252 are all temperature-sensitive mutants that cause cellular lethality at high temperatures, only dnaB107 stimulates tandem repeat recombination at its permissive temperature for growth. This may indicate that the other mutants function better than dnaB107 at their permissive temperatures, or it may indicate true differences in how these mutants affect replication. Further study will identify regions and particular functions of the DnaB protein that influence recombination. The dnaB107 deletion phenotype is unlikely to be due to a specific defect in priming, because at its permissive temperature a dnaB107 mutant synthesizes Okazaki fragments (LARK and WECHSLER 1975 ; SCLAFANI and WECHSLER 1981B ). Wild-type deletion rates were also exhibited by primase mutant dnaG2903. However, dnaG2903 was isolated as a chromosomal partitioning mutant and shows little defect in primer synthesis (VERSALOVIC and LUPSKI 1997 ). The wild-type deletion rate shown by initiation mutant dnaB252 (LANKA et al. 1978 ) suggests that the hyperdeletion phenotype conferred by dnaB107 is also unlikely to be due to a defect in replication initiation or interaction with DnaC. The dnaB252 mutant shows wild-type helicase and ATPase activity, but can be suppressed by overexpression of DnaC protein (SCLAFANI and WECHSLER 1981A ; SALUGA and GODSON 1995 ). It is speculated to be defective in DnaC-DnaB interactions that are required to load the helicase onto DNA templates. Strains carrying an ectopic terB site that blocks DnaB helicase progression prematurely on the E. coli chromosome are similar to dnaB107 strains in exhibiting hyperrecombination and viability dependent on RecB (HORIUCHI and FUJIMURA 1995 ). We believe that dnaB107 mutants most likely are specifically defective in their helicase activity and produce stalled replication forks even at their permissive temperature for growth. The hyperdeletion phenotype could then be a result of the breakage and subsequent repair of arrested replication forks. Further characterization of the DnaB107 protein should provide insight into its role in recombination events associated with the replication fork.

	ACKNOWLEDGMENTS

We are indebted to the following individuals for providing strains: R. Britton, R. Lloyd, R. Maurer, B. Michel, S. Sandler, and N. Sargentini. We thank Rachel Aubuchon and Vincent Sutera for construction of pSTL265. This work was supported by Public Health Service grants T32 GM07122 (to C.J.S.) and RO1 GM51753.

Manuscript received August 27, 1998; Accepted for publication February 2, 1999.

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