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Genetics, Vol. 166, 1631-1640, April 2004, Copyright © 2004

RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Janet R. Donaldsona, Charmain T. Courcellea, and Justin Courcellea
a Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi 39762

Corresponding author: Janet R. Donaldson, Mississippi State University, Box GY, Mississippi State, MS 39762., jrp12{at}msstate.edu (E-mail)

Communicating editor: G. R. SMITH


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

ALL cells must accurately replicate their entire genome each time they reproduce. Although the replication machinery is extremely processive, DNA damage such as that induced by near-ultraviolet light (254 nm) can block the progression of the DNA replication machinery and prevent it from completing its task (SETLOW et al. 1963 Down; HOWARD-FLANDERS et al. 1968 Down). The failure to accurately resume replication following disruption by DNA damage can result in mutation if an incorrect nucleotide is incorporated, rearrangement if replication resumes from the wrong site, or lethality if the blocking lesions cannot be overcome. In Escherichia coli, the recovery of replication following UV irradiation correlates with the time at which the lesions have been repaired by nucleotide excision repair (COURCELLE et al. 1999 Down, COURCELLE et al. 2003 Down). Cells deficient in nucleotide excision repair are unable to remove UV-induced DNA lesions, fail to recover replication, and exhibit elevated levels of mutagenesis, rearrangements, and cell lethality (HOWARD-FLANDERS 1968 Down; HOWARD-FLANDERS et al. 1968 Down; ROTHMAN and CLARK 1977 Down; COURCELLE and HANAWALT 2001 Down; HANAWALT 2002 Down).

The recovery of replication also depends on RecA and several gene products of the RecF pathway (ROTHMAN and CLARK 1977 Down; COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down; COURCELLE and HANAWALT 2001 Down). In the absence of RecA, RecF, RecO, or RecR, the blocked replication fork is not maintained, replication fails to recover, and extensive degradation of the nascent DNA at the replication fork occurs (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down, COURCELLE et al. 2003 Down; CHOW and COURCELLE 2004 Down). The RecQ helicase and RecJ nuclease also belong to the RecF pathway and partially degrade the nascent lagging strand of the arrested replication fork prior to the resumption of replication (COURCELLE and HANAWALT 1999 Down; COURCELLE et al. 2003 Down). These observations have led to the general model that RecA and these RecF pathway gene products function to maintain and process blocked replication forks until the blocking lesion can be repaired by nucleotide excision repair or bypassed by translesion DNA polymerases (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down, COURCELLE et al. 2003 Down; RANGARAJAN et al. 2002 Down). It is proposed that RecF, RecO, and RecR help stabilize activated RecA filaments at the arrested replication fork structure, thereby maintaining the replication fork DNA and limiting the degradation of the nascent DNA by RecJ and RecQ (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down, COURCELLE et al. 2003 Down; CHOW and COURCELLE 2004 Down). The genetic observation that mutations in recF, recO, or recR delay the induction of LexA-regulated gene expression following DNA damage is consistent with the idea that there is less activated RecA present at early times when RecF-O-R is absent (THOMS and WACKERNAGEL 1987 Down; HEGDE et al. 1995 Down).

On the basis of this model, it has been proposed that the repair of the DNA lesions in this situation may require displacement of the arrested replication machinery and nascent DNA to allow repair enzymes to gain access to the damaged region (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down, COURCELLE et al. 2001 Down). The displacement of the nascent DNA would allow the parental template strands in that region to reanneal, in effect reversing the branch point of the replication fork to generate a four-arm regressed intermediate (Fig 1). Other models have speculated that replication fork regression could facilitate a recombination-mediated template switch that allows synthesis to occur past the blocking lesion (HIGGINS et al. 1976 Down; KUZMINOV 2001 Down; MICHEL et al. 2001 Down; COX 2002 Down; LUSETTI and COX 2002 Down; JAKTAJI and LLOYD 2003 Down; WEST 2003 Down).



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  		Figure 1. Proposed function of RecG or RuvAB during the recovery of replication forks arrested at UV-induced lesions. (A) Replication is normally extremely processive (B) but is arrested by DNA lesions in the leading strand template (HIGUCHI et al. 2003 Down; PAGES and FUCHS 2003 Down). (C) RecQ displaces the nascent lagging strand for degradation by RecJ. On the basis of in vitro characterizations, it has been suggested that RecG or RuvAB catalyze fork regression as part of the process required for the recovery of replication. (D) RecFOR and RecA limit nascent DNA degradation and maintain the replication fork until the blocking lesions can be repaired or bypassed. (E) Then the replication fork can be reestablished and (F) processive replication can resume.

Direct evidence for regressed replication fork intermediates has been observed following replication arrest on plasmids. Plasmid replication forks blocked by the DNA-binding protein Tus form a reversed intermediate both in vivo and in vitro (POSTOW et al. 2001 Down; OLAVARRIETA et al. 2002 Down). In this case, replication fork regression occurs spontaneously following arrest due to the unwinding of positive supercoils ahead of the replication fork (POSTOW et al. 2001 Down). A transient regression of the replication fork also occurs following arrest by UV-induced DNA damage on plasmids in vivo (COURCELLE et al. 2003 Down). The regressed replication fork persists until a time correlating with lesion removal and the resumption of DNA replication. Similar to arrested replication forks on the chromosome, the arrested replication fork intermediates on the plasmid are maintained by RecA, RecF, RecO, and RecR and are processed by RecQ and RecJ (COURCELLE et al. 2003 Down).

Although UV-induced replication fork reversal occurs on plasmids, it is not known whether fork regression also occurs on the bacterial chromosome or whether fork regression is required for replication to resume following disruption. Both RecG and RuvAB have been proposed to catalyze fork reversal in vivo on the basis of their in vitro activities (COURCELLE et al. 2001 Down; MCGLYNN and LLOYD 2001A Down, MCGLYNN and LLOYD 2001B Down). The ruv locus, consisting of ruvA, ruvB, and ruvC, was originally identified in a genetic screen for UV-sensitive mutants (OTSUJI et al. 1974 Down). In addition to their hypersensitivity to UV, ruv mutants also exhibit lower recombination frequencies during conjugation and transduction and abnormalities in cell division following UV irradiation as seen by the accumulation of long filamentous cells that fail to undergo septation (OTSUJI et al. 1974 Down; LLOYD et al. 1984 Down). Purified RuvA and RuvB form a complex that binds to Holliday junctions and promotes ATP-dependent branch migration (PARSONS et al. 1992 Down; PARSONS and WEST 1993 Down). RuvC interacts with RuvAB at Holliday junctions and produces symmetric endonucleolytic incisions at the crossover point to resolve joint molecules (CONNOLLY et al. 1991 Down). In vitro, RuvAB can promote branch migration on synthetic replication fork structures to form Holliday junctions (MCGLYNN and LLOYD 2001A Down). However, the enzyme complex preferentially catalyzes the reverse reaction, converting a four-arm Holliday junction into a three-arm, replication fork-like structure (MCGLYNN and LLOYD 2001A Down).

Mutations that inactivate RecG also render cells moderately sensitive to UV and reduce the frequency of conjugational recombination (STORM et al. 1971 Down). Purified RecG is a helicase that is also capable of promoting branch migration of Holliday junctions (LLOYD and SHARPLES 1993 Down). In addition, RecG catalyzes the conversion of synthetic three-arm replication fork substrates into four-arm molecules in a manner that preferentially displaces what would represent the nascent lagging strand (MCGLYNN and LLOYD 1999 Down, MCGLYNN and LLOYD 2001B Down).

These biochemical characterizations have led to the general view that RecG and potentially RuvAB are required for the recovery of replication following UV-induced DNA damage. We examined this possibility directly and observed that following replication disruption by UV-induced DNA damage, the replication fork is maintained and DNA synthesis resumes at a time comparable to that of wild type when either RecG or RuvAB is absent.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Bacterial strains and UV irradiation:
Our parental strain, SR108, is a thyA36 deoC2 derivative of W3110 (DE LUCIA and CAIRNS 1969 Down). The strains HL946 (SR108 recF332::Tn3) and HL921 (SR108 recA306::Tn10) have been described previously (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down). The strains CL008 (SR108 recG258::Tn5), CL532 (SR108 ruvA59::Tn10), and CL578 (SR108 ruvAB6204::kan) were constructed by P1 transduction of the recG258::Tn5, ruvA59::Tn10, and ruvAB6204::kan alleles from JC19245 (gift from S. Sandler), RDK2641 (LOMBARDO and ROSENBERG 2000 Down), and TP541 (MURPHY et al. 2000 Down), respectively, into SR108. The strains CL628 (SR108 recQ6215::cam recF332::Tn3), CL011 (SR108 recG258::Tn5 recF332::Tn3), and CL744 (SR108 ruvAB6204::kan recF332::Tn3) were constructed by P1 transduction of the recQ6215::cam, recG258::Tn5, and ruvAB6204::kan alleles from TP648 (MURPHY et al. 2000 Down), CL008, and CL578, respectively, into HL946. The strain CL561 (SR108 recG258::Tn5 ruvA59::Tn10) was constructed by P1 transduction of the recG258::Tn5 allele into CL532. ruvA59::Tn10 is reported to be a polar mutation affecting both ruvA and ruvB (SHARPLES et al. 1990 Down). Phenotypes were confirmed by antibiotic resistance and, when appropriate, UV hypersensitivity or nascent DNA degradation. UV irradiation for all experiments was performed using a Sylvania 15-W germicidal lamp (254 nm) at an incident dose of 0.9 J/m2/sec.

UV survival studies:
Fresh overnight cultures were diluted 1:100 in 10 ml of Davis medium (2.0 g KH2PO4, 7.0 g K2HPO4, 0.5 g Na3C6H5O7, 0.1 g MgSO4, 1.0 g (NH4)2SO4 per liter, pH 7.0) supplemented with 0.4% glucose, 0.2% casamino acids, and 10 µg/ml thymine (DGCthy medium) and grown to an OD600 of 0.5 in a 37° shaking incubator. Serial dilutions of each culture were plated in triplicate on Luria-Bertani plates supplemented with 10 µg/ml thymine and UV irradiated at the indicated doses. Plates were incubated overnight at 37° and colonies were counted the next day.

Growth rates:
Fresh overnight cultures were diluted 1:1000 in DGCthy medium and 200-µl aliquots were plated on a 96-well microtiter plate. The OD600 for each culture was measured with Molecular Devices (Menlo Park, CA) SPECTRAmax Plus and analyzed with SOFTmax Pro 4.0.

Total DNA accumulation:
Fresh overnight cultures were diluted 1:100 in 40 ml DGCthy medium supplemented with 0.1 µCi/ml [3H]thymine (60.5 Ci/mmol) and grown to an OD600 of 0.4 in a 37° shaking incubator. At this time, half the culture was UV irradiated with 27 J/m2 and the other half was mock irradiated. At 5 min intervals, duplicate 200-µl aliquots were precipitated in 5 ml of 5% trichloroacetic acid (TCA) and filtered onto Millipore glass fiber prefilters. The amount of 3H-labeled DNA on each filter was determined by liquid scintillation counting (COURCELLE et al. 1999 Down).

Density labeling and CsCl analysis:
Fresh overnight cultures were diluted 1:100 in 20 ml of DGCthy medium supplemented with 0.1 µCi/ml of [14C]thymine (53 mCi/mmol) and were grown to an OD600 of 0.5 (~108 cells/ml) in a 37° shaking incubator. At this time, half the culture was UV irradiated with 27 J/m2 and the other half was mock irradiated. Cultures were then filtered onto FisherBrand general filtration 0.45-µm membranes, washed with NET buffer (10 mM NaCl, 10 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0), resuspended in 10 ml DGC medium supplemented with 20 µg/ml 5-bromouracil in place of thymine and 0.5 µCi/ml [3H]thymine (60.5 Ci/mmol), and allowed to recover for a period of 1 hr in a 37° shaking incubator. Two volumes of ice-cold NET buffer were added to the 10-ml cultures, and the cells were then pelleted, resuspended in 150 µl TE (10 mM Tris, 1 mM EDTA, pH 8.0), and lysed in 170 µl of 0.5 M H2KPO4/KOH, pH 12.5, and 1.25% Sarkosyl. Isopycnic alkali CsCl gradients composed of 0.3 g of a DNA lysate solution, 2.23 g CsCl, and 3.31 g of a 0.1 M H2KPO4/KOH, pH 12.5, solution (refractive index 1.4055) were centrifuged to equilibrium at 80,000 x g for 96 hr at 20°. Gradients were collected in ~30 fractions onto Whatman no. 17 paper, washed in 5% TCA, and then washed in 95% ethanol. The quantity of 3H and 14C in each fraction was determined by liquid scintillation counting (COURCELLE et al. 1997 Down).

Rate of DNA synthesis:
The assay to measure the rate of DNA synthesis was modified from previous studies (KHIDHIR et al. 1985 Down; RANGARAJAN et al. 2002 Down). Fresh overnight cultures were diluted 1:100 in 50 ml of DGCthy medium supplemented with 0.1 µCi/ml of [14C]thymine (53 mCi/mmol) and grown to an OD600 of precisely 0.3 in a 37° shaking incubator. At this time, half the culture was UV irradiated with 27 J/m2 and the other half was mock irradiated. At the indicated times, 1 µCi/ml [3H]thymidine (77.8 Ci/mmol) was added to duplicate 0.5-ml aliquots for 2 min at 37°, before the cells were lysed and DNA precipitated in 5 ml of 5% TCA and filtered onto Millipore glass fiber prefilters. The amount of 3H and 14C on each filter was determined by liquid scintillation counting.

Degradation of nascent and genomic DNA:
Fresh overnight cultures were diluted 1:100 in 10 ml DGCthy medium supplemented with 0.1 µCi/ml [14C]thymine (53 mCi/mmol) and grown to an OD600 of 0.4 in a 37° shaking incubator. Cultures were labeled for 5 sec with 1 µCi/ml [3H]thymidine (77.8 Ci/mmol), filtered onto FisherBrand general filtration 0.45-µm membranes, washed with NET buffer, and resuspended in nonradioactive DGCthy medium. Cultures were immediately irradiated with a UV dose of 27 J/m2. At the indicated times, duplicate 0.2-ml aliquots (triplicate for the 0 time point) were precipitated in 5 ml of 5% cold TCA and filtered on Millipore glass fiber prefilters. The amount of 3H and 14C on each filter was determined by liquid scintillation counting (COURCELLE et al. 1997 Down).


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

RuvAB and RecG are not required for the recovery of DNA synthesis following UV-induced DNA damage:
Isogenic strains lacking RecG, RuvAB, or both gene products were constructed by standard P1 transduction. As previously reported, the recG and ruvAB mutants were moderately hypersensitive to UV irradiation (Fig 2A; RYDER et al. 1994 Down; BOLT and LLOYD 2002 Down; GREGG et al. 2002 Down). Hypersensitivity was more severe in the ruvAB recG double mutant than in either single mutant and was comparable to that of recA (Fig 2A). However, unlike recA, the ruvAB recG double mutant grew poorly even in the absence of exogenous DNA damage (Fig 2B). Previous studies have documented that RecA is absolutely required for the recovery of replication following inhibition of DNA synthesis (HOWARD-FLANDERS 1968 Down), yet recA mutants grow at rates comparable to wild-type cells in the absence of DNA damage. This observation then suggests that replication is not frequently inhibited in the absence of DNA damage. Furthermore, the poor growth of the ruvAB recG mutant relative to that of recA under these same conditions indicates that RuvAB or RecG is required to process DNA structures other than disrupted replication forks that arise during the normal replication cycle. However, this observation alone does not preclude the possibility that, in addition to these alternative roles, they may also be required to process replication forks prior to their recovery.



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  		Figure 2. Survival following UV irradiation and growth in the absence of DNA damage of wild-type, recF, recG, ruvAB, and ruvAB recG strains of E. coli. (A) The percentage of cells surviving the indicated dose of UV irradiation is plotted for each strain. Survival curves represent an average of at least two independent experiments. (B) The OD600 of each strain is plotted over time. {blacksquare}, wild-type; {diamondsuit}, recF; {triangleup}, recG; {circ}, ruvAB; •, recA; and {square}, ruvAB recG.

To determine whether the hypersensitivity of ruvAB or recG mutants results directly from a failure to resume DNA synthesis following disruption by UV irradiation, we monitored DNA synthesis after UV irradiation in these mutants by [3H]thymine incorporation. Following a UV dose of 27 J/m2, wild-type cultures exhibited a transient arrest of replication before synthesis resumed at a rate comparable to that in unirradiated cultures (Fig 3). In contrast, recF mutants, which are deficient in the resumption of disrupted replication forks (COURCELLE et al. 1997 Down, COURCELLE et al. 1999 Down), exhibited no further increase in the amount of 3H-labeled DNA following UV irradiation. When we examined UV-irradiated cultures of ruvAB or recG, we observed that both mutants resumed replication at a time comparable to that in wild-type cultures (Fig 3).



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  		Figure 3. recG and ruvAB recover robust replication at a time similar to that in wild-type cells. Cultures grown in the presence of [3H]thymine were either UV irradiated with 27 J/m2 (•) or mock irradiated ({circ}). The amount of 3H incorporated over time is plotted. Cultures were irradiated at time 0. Each graph represents an average of three independent experiments. Error bars represent 1 SD.

We also examined ruvAB recG double mutants to determine if the absence of both gene products prevented the recovery of replication following UV-induced DNA damage. In these mutants, the rate of DNA synthesis recovered to an extent that was comparable to unirradiated ruvAB recG cultures. However, the slow growth that occurs in unirradiated ruvAB recG cultures makes it inappropriate to compare the recovery observed in this mutant directly to wild-type cells.

The recovery of replication in ruvAB and recG mutants was also monitored by density labeling the DNA synthesized during the first hour following UV irradiation. Irradiated or mock-irradiated cultures were incubated in medium containing 5-bromouracil in place of thymine for 1 hr such that the density of the DNA made during this period was greater than that of the DNA synthesized before treatment. DNA synthesized during the recovery period was then isolated and quantitated in isopycnic alkali CsCl gradients. By this measure, wild-type cultures had almost completely recovered replication 1 hr after UV irradiation, as seen by the nearly equivalent amounts of DNA synthesis in the irradiated and unirradiated cultures (Fig 4). By contrast, very little DNA synthesis occurred following UV treatment in recF mutants. When we examined postirradiation DNA synthesis in ruvAB and recG mutants, we observed an amount of DNA synthesis that was comparable to the unirradiated controls, indicating that DNA synthesis was resuming similar to that in wild-type cultures (Fig 4).



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  		Figure 4. recG and ruvAB cultures synthesize an amount of DNA similar to that of wild-type cultures during the first hour after UV irradiation. The amount of DNA synthesized in UV-irradiated (25 J/m2) or mock-irradiated cultures was determined by density labeling the DNA with 5-bromouracil and subsequent isolation in alkali CsCl gradients. {circ}, DNA synthesized before treatment (14C); {blacksquare}, DNA synthesized following treatment (3H). Each graph represents one of at least two independent experiments.

In ruvAB recG double mutants, we observed an intermediate amount of DNA synthesis in the irradiated culture relative to the unirradiated culture. However, both irradiated and nonirradiated cultures exhibited abnormal patterns of replication, with a significant amount of the DNA synthesis migrating at densities in the intermediate and light regions of the gradient. DNA migrating in these regions may indicate elevated levels of recombination or repair synthesis. The detection of this type of synthesis in unirradiated ruvAB recG mutants may be due in part to the toxicity associated with the 5-bromouracil that is used to density label the DNA in this assay. The toxicity of 5-bromouracil is thought to be due in part to the lower incorporation efficiency of this base analogue compared to thymine and also because the bromine group on the analogue is labile, leading to elevated levels of uracil and uracil glycolyase-induced nicks in the DNA. Incubation in media containing 5-bromouracil results in elevated levels of sister chromatid exchanges and cell death within approximately two rounds of replication (HACKETT and HANAWALT 1966 Down; LITTLE 1976 Down; KRASIN and HUTCHINSON 1978 Down). Thus, similar to the previous assay, a direct comparison between wild-type and ruvAB recG mutants should be interpreted with caution. However, some DNA synthesis occurs in the UV-irradiated ruvAB recG mutants, although the viability of these cells is clearly compromised and abnormal relative to wild-type cells even in the absence of UV irradiation.

The previous two assays indicate that replication recovers in the absence of either recG or ruvAB. However, it remains possible that although robust replication resumes in ruvAB or recG mutants, the time at which DNA synthesis recovers may be delayed relative to that of wild type. To examine this possibility in recG and ruvAB mutants, we measured the rate of DNA synthesis following UV irradiation by incubating [14C]thymine-labeled cultures for 2 min with [3H]thymidine at various times after treatment. The rate of DNA synthesis (3H incorporation/min) could then be determined relative to the total amount of DNA present (14C incorporation) at specific times following treatment. Using this assay, we observed that the rate of DNA synthesis was reduced by ~90% in wild-type cells at early times following UV irradiation (Fig 5). Within 20 min, the rate of DNA synthesis began to recover, and by 40 min, the rate of replication was nearly restored to preirradiation levels and there was a detectable increase in total DNA accumulation. In UV-irradiated recF mutants, the reduction in DNA synthesis was more severe and, consistent with our previous assays, the rate of synthesis did not recover. However, following UV irradiation of recG or ruvAB mutants, we observed that the time and efficiency with which DNA synthesis recovered were similar to those in wild type. These observations indicate that RuvAB or RecG function is not essential for replication to resume following disruption by UV-induced DNA damage. In the ruvAB recG double mutants, the rate of DNA synthesis recovered to a significant extent and approximated the recovery observed in wild-type cultures much more closely than that observed in recF mutants. Although direct comparisons between these strains should be made with caution, the observation that DNA synthesis is inhibited to a greater extent in recF mutants than in ruvAB recG double mutants suggests that the recovery of DNA synthesis in the single mutants is not due to the simple interpretation that RecG and RuvAB serve redundant functions in this respect. The double mutant recovers to a greater extent than the recF mutant despite the fact that it is much more sensitive to DNA damage and grows more poorly than the recF mutant (Fig 2).



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  		Figure 5. recG and ruvAB resume DNA synthesis with kinetics similar to that of wild-type following UV irradiation. Cultures grown in the presence of [14C]thymine were pulse-labeled with [3H]thymidine for 2 min at the indicated times following either 27 J/m2 of UV irradiation or mock irradiation. The relative amount of 14C and 3H incorporated into the DNA is plotted over time. Cultures were irradiated at time 0. Graphs represent an average of three independent experiments. Error bars represent 1 SD. {circ}, total DNA in mock-irradiated cultures; •, total DNA in irradiated cultures; {square}, rate of DNA synthesis in mock-irradiated cultures; {blacksquare}, rate of DNA synthesis in irradiated cultures.

RuvAB and RecG are not required to maintain the replication fork after UV irradiation:
Strains lacking RecF, RecO, or RecR fail to maintain disrupted replication forks, resulting in extensive degradation of the nascent DNA at the replication fork (COURCELLE et al. 1997 Down, COURCELLE et al. 2003 Down). Both RecG and RuvAB have also been proposed to act on arrested replication fork structures in vivo (MCGLYNN and LLOYD 2001A Down, MCGLYNN and LLOYD 2001B Down). To determine whether RuvAB or RecG are required to maintain replication forks arrested at UV-induced DNA damage in vivo, we measured the amount of degradation that occurred in the nascent DNA at the replication fork. To this end, 14C-labeled cultures were pulse labeled with [3H]thymidine for 5 sec, transferred to nonradioactive media, and immediately UV irradiated. Then, the amount of radioactivity remaining in the DNA was followed over time. This assay allowed us to compare the amount of degradation that occurred in the nascent strands of the replication fork directly to the total DNA in the cell. In UV-irradiated wild-type cells, we observed a limited amount of nascent DNA degradation at times prior to the recovery of replication, consistent with our previous studies (Fig 6; COURCELLE and HANAWALT 1999 Down). In recF mutants, the replication fork was not maintained and approximately half of the nascent DNA was degraded. By comparison, in ruvAB or recG mutants, the nascent DNA was not extensively degraded following UV irradiation. In addition, ruvAB recG double mutants did not exhibit extensive degradation of DNA following UV irradiation, indicating that these gene products are not required to maintain or protect the nascent DNA at replication forks.



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  		Figure 6. RuvAB or RecG is not required to maintain arrested replication forks or displace the nascent DNA prior to degradation following UV irradiation. [3H]Thymidine was added to [14C]thymine prelabeled cells for 5 sec, washed, and UV irradiated in nonlabeled medium. The relative amount of 3H and 14C remaining in the DNA is plotted over time. Graphs represent an average of at least three independent experiments for each strain. Error bars represent 1 SD. {circ}, total DNA (14C); •, nascent DNA (3H).

The nascent DNA degradation that occurs prior to the resumption of replication is dependent on RecQ helicase and RecJ nuclease (COURCELLE and HANAWALT 1999 Down). RecQ helicase is required to displace the nascent lagging strand for degradation by RecJ nuclease in vivo. On the basis of in vitro characterizations, it has been proposed that RecG and potentially RuvAB also displace the nascent lagging strand of arrested replication forks (MCGLYNN et al. 2001 Down; MICHEL et al. 2001 Down). If true, then we would predict that inactivation of RecG or RuvAB should also prevent nascent DNA degradation from occurring similar to when RecQ is inactivated. To test this possibility, we examined the degradation that occurred in UV-irradiated recF mutants that were also deficient in either RuvAB or RecG. As shown in Fig 6, the absence of RuvAB or RecG did not prevent the extensive degradation of the nascent DNA in recF mutants. In contrast, in recF mutants that also lacked RecQ, the degradation of nascent DNA was significantly reduced. The lack of nascent DNA processing in the recF recQ mutant was most evident during the first hour following UV irradiation (Fig 6). This result indicates that RuvAB and RecG are not required to displace the nascent lagging strand prior to degradation in vivo.

Since this assay specifically measures nascent DNA degradation, and previous studies have shown this degradation occurs preferentially on the nascent lagging strand (COURCELLE and HANAWALT 1999 Down), it remains possible that RuvAB or RecG facilitates the displacement of the nascent leading strand or portions of the lagging strand that are not subject to degradation and therefore are not detected in this assay.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

On the basis of biochemical data, several studies have speculated that either RecG or RuvAB catalyze replication fork regression in vivo and play a critical role in promoting the recovery of replication when it is blocked by DNA damage (MCGLYNN and LLOYD 1999 Down, MCGLYNN and LLOYD 2001A Down, MCGLYNN and LLOYD 2001B Down; BOLT and LLOYD 2002 Down; GREGG et al. 2002 Down). Using a number of cellular assays, we examined the contribution of RuvAB and RecG to the ability of cells to recover replication following UV irradiation in vivo. We found that the absence of either RecG or RuvAB does not affect the cell's ability to resume DNA synthesis. In addition, unlike RecF, RecO, or RecR, we observe that RuvAB or RecG is not required to maintain replication forks following arrest by DNA damage and that neither protein prevents the extensive nascent DNA degradation that occurs in the absence of RecF.

Although these results cannot exclude the possibility that RuvAB or RecG proteins catalyze fork regression in vivo, they demonstrate that their function is not required for DNA synthesis to resume following UV-induced DNA damage. It remains possible that RuvAB- or RecG-catalyzed replication fork regression increases the accuracy or fidelity of replication recovery, but that the regression is not essential for the resumption to occur. By analogy, both RecJ and RecQ process or partially degrade the nascent DNA at arrested replication forks in a manner that is believed to increase the frequency that replication resumes from the proper location (COURCELLE and HANAWALT 1999 Down). However, the absence of either RecJ or RecQ does not prevent replication from resuming following UV irradiation, although it does affect the time at which DNA synthesis resumes (COURCELLE and HANAWALT 1999 Down). A second possibility is that fork regression catalyzed by RecG and RuvAB is required for recovery in only a small subset of the total arrested-fork substrates and therefore is below our limit of detection in these cellular assays. Another possibility is that alternative or redundant activities may allow replication to recover in the absence of RecG or RuvAB. Along this line of reasoning, RadA was recently proposed to offer a third potentially redundant activity for replication fork processing on the basis of survival studies (BEAM et al. 2002 Down). However, if either RecG- or RuvAB-catalyzed fork regression is a predominant mechanism by which arrested replication forks normally resume, it seems reasonable to expect that we would have observed a delay in the timing, or a reduction in the efficiency, of the resumption of DNA synthesis. Even allowing for potential redundancies, one might expect that the secondary activity would promote recovery with different (or reduced) kinetics when the primary activity is absent. Our observations show that even though ruvAB and recG mutants are more sensitive to UV irradiation than wild-type cells are, ruvAB and recG mutants are able to recover DNA synthesis as efficiently as wild-type cells, arguing against a requirement for either of these enzymes in a prominent pathway that allows DNA synthesis to resume.

The poor growth of ruvAB recG double mutants is often interpreted to suggest that replication is frequently disrupted by DNA damage or other impediments during replication, which then requires processing by branch migration enzymes to resume (MANDAL et al. 1993 Down; RYDER et al. 1994 Down). However, cell viability is an extremely broad criterion by which to measure a specific question such as the ability for DNA synthesis to resume. The survival of a cell could also be compromised by any of a large number of alternative DNA processing events such as chromosome partitioning, replication termination, or resolution of joint dimer chromosomes, among others. The observation that wild-type cells recover from UV doses that reduce the viability of recG or ruvAB mutants by >99% highlights the observation that these enzymes are essential for some DNA processing event that arises in these cells following moderate levels of DNA damage. If any of the several processing events mentioned above were to function as the preferred targets for these branch migration enzymes, the normal resumption of DNA synthesis would not be expected to be impaired, but could result in elevated levels of lethality in the presence of DNA damage. A second possibility is that the DNA synthesis that occurs in recG or ruvAB mutants represents an aberrant form of DNA synthesis, potentially resuming from the wrong template, and leads to lethality in the absence of RecG or RuvAB processing. However, it is clear from the observations presented in this study that the lethality is not the result of a failure to resume DNA synthesis, such as occurs in recF or recA mutants.

Other genetic studies have previously been interpreted to support a role for RuvAB or RecG at arrested replication forks. Following prolonged incubation of a thermosensitive dnaB mutant at the restrictive temperature, elevated levels of double-strand breaks accumulate in the genome of recBC mutants as observed by pulsed-field gel electrophoresis (MICHEL et al. 1997 Down). The accumulation of double-strand breaks in dnaB recB mutants requires RuvABC function (SEIGNEUR et al. 1998 Down). On the basis of these observations, it has been speculated by others that inactivation of replication proteins such as DnaB may mimic the disruption that occurs following replication fork encounters with DNA damage (GREGG et al. 2002 Down; JAKTAJI and LLOYD 2003 Down). It was further speculated that if this interpretation were true, then the RuvAB-dependent double-strand breaks could be explained if RuvAB catalyzed the formation of Holliday junctions at stalled replication forks, which are then cleaved by RuvC endonuclease or degraded by RecBCD (COX et al. 2000 Down; BOLT and LLOYD 2002 Down; GREGG et al. 2002 Down). On the basis of these observations in thermosensitive replication mutants, it has been inferred from several subsequent studies that RuvABC and RecBCD are required to resume replication following arrest by DNA damage. However, our observations indicate that the resumption of DNA synthesis following UV-induced DNA damage does not require RuvAB, and several previous studies have shown that replication resumes normally in recBC and recD mutants following UV-induced DNA damage (KHIDHIR et al. 1985 Down; COURCELLE et al. 1997 Down; COURCELLE and HANAWALT 1999 Down). Furthermore, although RecJ and RecQ process the nascent DNA at lesion-arrested replication forks, RecBCD does not degrade the nascent DNA at arrested replication forks (KHIDHIR et al. 1985 Down; COURCELLE et al. 1997 Down; COURCELLE and HANAWALT 1999 Down). Therefore, we believe these observations indicate that the events and enzymes operating at lesion-blocked replication forks are different from those that occur following the removal of specific proteins of the replication machinery.

The basis for the proposal that RecG may promote the rescue of arrested replication forks in vivo comes primarily from survival studies following UV irradiation (GREGG et al. 2002 Down; JAKTAJI and LLOYD 2003 Down). By examining the survival of recG mutants following UV irradiation in various genetic backgrounds, it has been widely speculated that RecG promotes the rescue of stalled replication forks through a number of different recovery pathways (MCGLYNN and LLOYD 2000 Down, MCGLYNN and LLOYD 2002 Down; DILLINGHAM and KOWALCZYKOWSKI 2001 Down; GREGG et al. 2002 Down; JAKTAJI and LLOYD 2003 Down). However, if this were true, one would predict that the absence of RecG would have an effect on the cell's ability to recover DNA synthesis following UV irradiation. Our observation that recG recovers DNA synthesis with kinetics similar to that in wild-type cells argues against the interpretation that RecG has an essential role in promoting the rescue of arrested replication forks following UV-induced DNA damage. However, this does not necessarily exclude the possibility that RecG participates in the recovery process or possibly enhances strand displacement at arrested replication forks.

Although many gene products have been intensely studied for how they affect recombinational processes over the years, the conceptual realization that many of the "rec" gene products function to maintain the strands of genetic information rather than rearrange them during chromosome replication has been suggested previously and investigated recently (CAMPBELL 1984 Down; COURCELLE et al. 1997 Down). This perspective has generated significant amounts of discussion and renewed enthusiasm concerning how replication recovers following disruption. As a result, in some cases, it has been speculated that genes are required for this process primarily on the basis of the observation that their respective mutants grow poorly or render cells hypersensitive to UV. RuvAB and RecG have both been characterized for their recombinational roles and only recently were proposed to promote the recovery of replication on the basis of the observation that the mutants are moderately hypersensitive to UV irradiation (MCGLYNN and LLOYD 2001A Down, MCGLYNN and LLOYD 2001B Down; MCGLYNN et al. 2001 Down; BOLT and LLOYD 2002 Down). Although this represents a good starting point for investigation, the recovery of replication is not the only process required for successful reproduction, and UV hypersensitivity does not always correlate with a defect in the recovery of replication. For example, both recF and recBC mutants are equally hypersensitive to UV irradiation, but while recF mutants fail to recover replication following UV irradiation, recBC mutants recover replication normally (COURCELLE et al. 1997 Down; COURCELLE and HANAWALT 1999 Down). By comparison, recJ and recQ mutants are not sensitive to UV irradiation, yet these gene products participate in the processing of nascent DNA at replication forks prior to the recovery of replication (COURCELLE and HANAWALT 1999 Down). Although the results presented here cannot exclude the possibility that RuvAB or RecG participate in the regression of the arrested replication forks, we can conclude that neither gene product is essential for DNA synthesis to resume. This implies that the hypersensitivity of these mutants arises from an event(s) that potentially occurs after replication resumes or because replication resumes from the wrong template or the wrong place in their absence. It will be important to develop novel cellular assays to determine whether replication fork regression is required for the resumption of replication on the bacterial chromosome. In light of these observations, it may also be useful to consider potential alternative roles for RecG and RuvAB function following UV-induced DNA damage.


*  ACKNOWLEDGMENTS

We thank Anthony Poteete, Susan Rosenberg, and Steve Sandler for providing bacterial strains used in this study. This work was supported by award MCB0130486 from the National Science Foundation and a National Research Service Award F32 GM-068566 from the National Institutes of Health to C.T.C.

Manuscript received November 26, 2003; Accepted for publication January 6, 2004.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
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