JOURNAL OF BACTERIOLOGY, Nov. 2005, p. 76077618 0021-9193/05/$08.00 0 doi:10.1128/JB.187.22.76077618.2005 Copyright 2005, American Society for Microbiology.

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Vol. 187, No. 22

Roles of the Escherichia coli RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In Vivo
Robert W. Maul and Mark D. Sutton*
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214
Received 8 July 2005/Accepted 25 August 2005

The Escherichia coli sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the dnaN159 allele, which encodes a mutant form of the clamp ( 159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:67386748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the ve E. coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the dinB-encoded polymerase IV is sufcient to result in conditional lethality of the dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., recR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., polB or umuDC). Thus, we have identied distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cells different DNA polymerases are managed in vivo. The nding that Escherichia coli possesses ve distinct DNA polymerases, while humans are currently known to possess at least three times this many (reviewed in references 15 and 61), suggests that organisms must employ a series of adaptable control systems in order to ensure that the correct DNA polymerase gains access to the replication fork at the appropriate time. To some extent, a cell can regulate the access to the replication fork of its different DNA polymerases by attenuating their respective expression levels, specic activities, and/or subcellular localizations (reviewed in reference 61). However, even with these control systems in place, situations will inevitably arise in which a cell must choose between multiple DNA polymerases. As selection of the incorrect polymerase could have catastrophic consequences, it seems likely that control systems in addition to those mentioned above must exist in order to ensure that in these situations, the correct DNA polymerase is recruited to the replication fork at the appropriate time. When considering how E. coli might manage the actions of its ve DNA polymerases to coordinately regulate DNA replication with DNA repair and translesion DNA synthesis, one cannot overstate the importance of the global SOS response: the steady-state levels of polymerase II (Pol II), Pol IV, and Pol V vary as much as 10-fold or more as a function of SOS induction (23). Proper management of the E. coli SOS response requires the products of the lexA and recA genes. LexA protein acts as a transcriptional repressor. By binding to sequences termed SOS boxes located near promoters, LexA
* Corresponding author. Mailing address: Department of Biochemistry, 140 Farber Hall, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214. Phone: (716) 829-3581. Fax: (716) 829-2661. E-mail: mdsutton@Buffalo .edu. 7607

blocks access of RNA polymerase to the promoter, thereby effectively repressing transcription of more than 40 different genes (10, 16). RecA protein, which is required for most homologous recombination (reviewed in reference 30), as well as for the repair of double-stranded DNA breaks (reviewed in references 24 and 30) and the restart of stalled replication forks (reviewed in reference 11), becomes activated for its role in SOS induction by binding to single-stranded DNA (ssDNA), which presumably results from the cells failed attempts to copy over lesions in the DNA (reviewed in references 16 and 60). Upon binding to ssDNA, RecA forms a helical lament structure, referred to as activated RecA (30). Interaction of LexA with activated RecA mediates autodigestion of LexA, which largely inactivates its repressor function, leading to transcriptional derepression of the LexA-regulated genes (reviewed in reference 16). Although many of the SOS-regulated genes, including polB (Pol II) and dinB (Pol IV), are expressed at modest levels in the absence of DNA damage, others, such as umuDC (Pol V), are expressed at easily detectable levels only following SOS induction (16, 60). Moreover, catalytic activity of Pol V requires that its umuD-encoded subunit undergo a RecA/ ssDNA-facilitated autodigestion that is mechanistically similar to that of LexA (7, 41, 45, 53): intact UmuD, together with UmuC, acts in a DNA damage checkpoint control (38, 43), while autodigestion of UmuD results in the removal of its N-terminal 24 residues to generate UmuD (7, 41, 53), which leads to the release of the checkpoint and concomitant activation of Pol V-dependent translesion DNA synthesis. In addition to its role in the expression and posttranscriptional activation of Pol V, RecA also plays an as yet poorly understood role in stimulating the catalytic activity of Pol V (46, 47, 49, 52, 61).

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MAUL AND SUTTON

J. BACTERIOL.

In addition to SOS regulation, work from a variety of laboratories (2, 5, 8, 12, 27, 29, 67), including our own (5659), indicates that the E. coli sliding clamp and the clamp loader complex play important roles in coordinating access of the different DNA polymerases to the replication fork. The clamp is a ring-shaped, homodimeric protein that topologically encircles double-stranded DNA. It is loaded onto DNA by the complex, which is comprised of six distinct polypeptides ( 2, , , , , and ) (6, 34). Although the mechanism by which the clamp is loaded onto DNA is not yet fully understood, it is clear that complex binds to the clamp in an ATPdependent fashion to catalyze the opening of the clamp. After positioning the opened clamp on the DNA so that it encircles double-stranded DNA, the complex undergoes a conformational change that is triggered by the ATPase activity of the and subunits, which effects release of the clamp, resulting in its loading (1, 20). Once loaded, the clamp moves freely along the DNA, acting as a mobile platform (55). All ve E. coli DNA polymerases interact with , and this interaction serves to tether the polymerases to the DNA, endowing them with various degrees of processivity (5, 29, 31, 33, 63, 67). In the case of Pol IV, this interaction also serves to enhance its afnity for deoxyribonucleotides (3). Moreover, it has recently become clear that all ve DNA polymerases bind to overlapping surfaces on the clamp (28, 69), suggesting that competition of the different DNA polymerases for interaction with the clamp may help to coordinate polymerase access to the replication fork (12, 28, 66, 67, 69). Consistent with the idea that the clamp and the clamp loader complex help to coordinate access to the replication fork of the different DNA polymerases, Viguera et al. (65) determined that a holD::kan mutation, which affects the subunit of the clamp loader complex, conferred a temperature-sensitive phenotype that was suppressed by deletion of polB, and to a lesser extent by deletion of dinB. Furthermore, we previously reported that the dnaN159 allele, which encodes a mutant form of the sliding clamp bearing G66E and G174A substitutions (19, 42, 56), was impaired for interaction with the catalytic subunit of Pol III and conferred a UV-sensitive phenotype in a nucleotide excision repair-decient background that was suppressed by (not epistatic with) inactivation of Pol IV (56). Taken together, these ndings suggest that mutations that impair either the clamp or the efciency of clamp loading lead to altered polymerase usage, consistent with a role for the clamp in helping to regulate Pol access to the replication fork. Presumably, the ability of the SOS-regulated DNA polymerases to impair growth of a dnaN159 or holD::kan strain is due in large part to a replication defect conferred by the mutations. Although it is possible that the various polymerases compete with each other for access to the replication fork in a stochastic manner, the fact that only particular SOS-regulated DNA polymerases impaired growth in specic genetic backgrounds suggested to us that additional factors, such as the steady-state levels of the different DNA polymerases, which are heavily inuenced by the SOS response, as well as other SOS-regulated gene products, could help regulate access of the SOS-regulated polymerases to the replication fork. To test this hypothesis, we used different lexA and recA alleles to determine the individual effects of SOS induction and RecA activation on polymerase usage in the dnaN159 mutant.

Our results indicate that elevated levels of Pol IV impaired growth of the dnaN159 mutant. However, the recA730 allele, which expresses a mutant form of the RecA protein (RecA730) that displays an enhanced ability to form RecA/ssDNA nucleoprotein laments, conferred a Pol II- and Pol V-dependent lethality in the dnaN159 mutant. Lethality conferred by Pol V did not correlate with its expression level, but rather was dependent upon recA730. Taken together, these results suggest that the LexA and RecA proteins play an important role in effecting polymerase selection and/or polymerase switching at the replication fork following DNA damage. These ndings are discussed in terms of a model to describe how E. coli manages the actions of its different DNA polymerases.
MATERIALS AND METHODS E. coli strains and bacteriological techniques. E. coli strains are described in Table 1 and were routinely grown in Luria-Bertani (LB) medium (36). When necessary, the following antibiotics were used at the indicated concentrations: ampicillin, 150 g/ml; chloramphenicol, 20 g/ml; kanamycin, 60 g/ml; spectinomycin, 50 g/ml; streptomycin, 50 g/ml; tetracycline, 10 g/ml; and rifampin, 100 g/ml. All strains are derivatives of E. coli K-12 and were constructed using P1vir-mediated generalized transduction (36). Transduction of the recA730 and recA441 alleles was achieved by selection for tetracycline resistance conferred by the closely linked srlC300::Tn10 allele, and the correct sequence for each allele was conrmed by automated nucleotide sequence analysis of the PCR-amplied recA gene. Transduction of the dnaN159 allele was achieved by selection for the closely linked tnaA300::Tn10 or zid-3162::Tn10kan alleles and was conrmed by verifying temperature sensitivity and by nucleotide sequence analysis of the PCR-amplied dnaN allele, as previously described (56). Transduction of the (araD-polB):: , (dinB-yafN)::kan, and umuDC595::cat alleles was done by virtue of antibiotic resistance conferred by the indicated marker. Due to the high background of spectinomycin-resistant CFU occasionally observed when selecting for strains bearing the fragment (regardless of the spectinomycin concentration used; data not shown), we transduced strains that we intended to make (araD-polB):: to tetracycline resistance with leu::Tn10, which is adjacent to polB, and then replaced leu::Tn10 with (araD-polB):: . The presence of each allele was subsequently conrmed by colony PCR. The (araD-polB):: allele was conrmed using primers 5 -CCG ACG GGA TCA ATC AGA AAG GTG-3 and 5 -TCT GTC CTG GCT GGG AAC GA-3 , which amplify a 817-base-pair fragment from (araD-polB):: and yield no product using the polB allele. The (dinB-yafN)::kan allele was conrmed using primers 5 -cgc gaa ttc cat ATG CGT AAA ATC ATT CAT GTG GAT ATG G-3 (the rst 12 bases bear no sequence similarity to the dinB gene; rather, they introduce an NdeI restriction site that was used for cloning dinB into an overexpression vector) and 5 -CCG GTT GAT CAA TAA AGT ATT TAG CTG GG-3 , which amplify a 1,000-base-pair fragment from the dinB -yafN region, and a 1,250-basepair fragment from (dinB-yafN)::kan. The umuDC595::cat allele was conrmed using primers 5 -AGG CCA CGT GAG CAC AAG ATA AGA-3 and 5 -ATA GGT ACA TTG AGC AAC TGA CTG-3 , which amplify a 530-basepair fragment from umuDC595::cat strains and yield no product using the umuD C alleles. Plasmid construction and transformation assay. Plasmid DNAs are described in Table 1. Genes encoding Pol I (polA), Pol II (polB), and Pol IV (dinB) were PCR amplied from genomic DNA, and a synthetic operon encoding Pol V (umuD C ) was PCR amplied from plasmid pGW3751 (41) using the following primer pairs: polA-promoter, 5 -CTT GCG TGA AAC GGG CGC CTT-3 and polA-end, 5 -ggg aca cct agg TTA GTG CGC CTG ATC CCA G-3 (the rst 12 nucleotides are not complementary to polA and were included for cloning purposes); polB-promoter, 5 -CAC TAT CTG CGT AAG CAT GGC GCG AAG GC-3 and polB-end, 5 -ggg aca cct agg TCA GAA TAG CCC AAG TTG C-3 (the rst 12 nucleotides are not complementary to polB and were included for cloning purposes); dinB-promoter, 5 -CAA TAA GAA TTC CGT CAA TCG CCA TCT GTT TGC CGG G-3 and dinB-end, 5 -cgc aca aag ctt ggt acc TCA TAA TCC CAG CAC CAG TTG TC-3 (the rst 18 nucleotides are not complementary to dinB); and umuDC-promoter, 5 -CTG CTG GCA AGA ACA GAC-3 and umuDC-end, 5 -CGT GAT CTG TTC GGT CGC TAA TCC-3 . PCR fragments were blunt-end ligated into pCR-BluntII-Topo vector (Invitrogen, Carlsbad, CA) as per the manufacturers recommendations. After verifying that each clone contained the correct nucleotide sequence (Roswell Park Cancer

VOL. 187, 2005

RecA AND DNA POLYMERASE SELECTION TABLE 1. E. coli strains and plasmids used in this studya

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Strain or plasmid Strain AM107 CAG18497 CAG18558 DE298 FC1237 GW8028 JJC1209 RW120 STL1336 MS100 MS101b RW542 MS104 MS105 RM100 RM101 RM102 RM103 RM104 RM105 RM106 RM107 RM108 RM109 RW540 RM110 RM111 RM112 RM113 RM114 RW576 RM115 RM116 RM117 RM118 RM119 RM120 RM121 RM122 RM123 RM124 RM125 RM126 RM127 RM128 RM129c RM130c RM131a RM132 RM133 RM134 RM135c RM136c Plasmids pWSK29 pRM100 pRM101 pRM102 pRM103

Relevant genotype recR252::Tn10 9kan fadR613::Tn10 zid-3162::Tn10kan recA441 srlC300::Tn10 (dinB-yafN)::kan ara-714 leu::Tn10 dnaN159 zid-3162::Tn10 umuDC595::cat (araD-polB):: tnaA300::Tn10 dnaN159 tnaA300::Tn10 lexA51(Def) lexA51(Def) tnaA300::Tn10 lexA51(Def) dnaN159 tnaA300::Tn10 lexA51(Def) (dinB-yafN)::kan lexA51(Def) umuDC595::cat lexA51(Def) ara-714 leu::Tn10 lexA51(Def) (araD-polB):: lexA51(Def) (araD-polB):: tnaA300::Tn10 lexA51(Def) (araD-polB):: dnaN159 tnaA300::Tn10 lexA51(Def) (dinB-yafN)::kan tnaA300::Tn10 lexA51(Def) (dinB-yafN)::kan dnaN159 tnaA300::Tn10 lexA51(Def) umuDC595::cat tnaA300::Tn10 lexA51(Def) umuDC595::cat dnaN159 tnaA300::Tn10 lexA51(Def) uvrA6 lexA51(Def) uvrA6 tnaA300::Tn10 lexA51(Def) uvrA6 dnaN159 tnaA300::Tn10 lexA51(Def) uvrA6 recA441 srlC300::Tn10 lexA51(Def) uvrA6 recA441 srlC300::Tn10 zid3162::Tn10kan lexA51(Def) uvrA6 recA441 srlC300::Tn10 dnaN159 zid-3162::Tn10kan lexA51(Def) uvrA6 recA730 lexA51(Def) uvrA6 recA730 tnaA300::Tn10 lexA51(Def) uvrA6 recA730 (dinB-yafN)::kan lexA51(Def) uvrA6 recA730 (dinB-yafN)::kan tnaA300::Tn10 lexA51(Def) uvrA6 recA730 umuDC595::cat lexA51(Def) uvrA6 recA730 umuDC595::cat tnaA300::Tn10 lexA51(Def) uvrA6 recA730 umuDC595::cat dnaN159 tnaA300::Tn10 lexA51(Def) uvrA6 recA730 ara-714 leu::Tn10 lexA51(Def) uvrA6 recA730 (araD-polB):: lexA51(Def) uvrA6 recA730 (araD-polB):: tnaA300::Tn10 lexA51(Def) uvrA6 recA730 (araD-polB):: dnaN159 tnaA300::Tn10 umuDC595::cat fadR613::Tn10 lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595::cat fadR613::Tn10 lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595::cat fadR613::Tn10 zid-3162::Tn10kan lexA51(Def) uvrA6 recA730 (araD-polB):: umuDC595::cat fadR613::Tn10 dnaN159 zid3162::Tn10kan lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-1 lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-2 lexA51(Def) uvrA6 recA730 dnaN159 tnaA300::Tn10 srd-1 umuDC595::cat lexA51(Def) uvrA6 recA730 recR252::Tn10 9kan lexA51(Def) uvrA6 recA730 recR252::Tn10 9kan tnaA300::Tn10 lexA51(Def) uvrA6 recA730 recR252::Tn10 9kan dnaN159 tnaA300::Tn10 lexA51(Def) uvrA6 recA730 zid-3162::Tn10kan srd-1 lexA51(Def) uvrA6 recA730 zid-3162::Tn10kan srd-2 Apr; Apr; Apr; Apr; Apr;

Source or construction J. Courcelle (9) CGSCb B. Michel (19) R. Woodgate P. Foster (54) G. Walker B. Michel (19) R. Woodgate (71) M. Goodman (4) This laboratory (56) This laboratory (56) R. Woodgate (21) This laboratory (56) This laboratory (56) P1(FC1237) RW542 P1(RW120) RW542 P1(GW8028) RW542 P1(STL1336) RM102 P1(MS100) RM103 P1(MS101) RM103 P1(MS100) RM100 P1(MS101) RM100 P1(MS100) RM101 P1(MS101) RM101 R. Woodgate P1(MS100) RW540 P1(MS101) RW540 P1(DE298) RW576 P1(CAG18558) RM112 P1(JJC1209) RM112 R. Woodgate P1(MS100) RW576 P1(FC1237) RW576 P1(MS100) RM116 P1(RW120) RW576 P1(MS100) RM118 P1(MS101) RM118 P1(GW8028) RW576 P1(STL1336) RM121 P1(MS100) RM122 P1(MS101) RM122 P1(CAG18497) RW120 P1(RM125) RM122 P1(CAG18558) RM126 P1(JJC1209) RM126 P1(MS101) RW576 P1(MS101) RW576 P1(RW120) RM129 P1(AM107) RW576 P1(MS100) RM132 P1(MS101) RM132 P1(CAG18558) RM129 P1(CAG18558) RM130

pSC101 derivative pWSK29 bearing polA (Pol I) pWSK29 bearing polB (Pol II) pWSK29 bearing dinB (Pol IV) pWSK29 bearing umuD and umuC (Pol V)

S. Kushner (68) This work This work This work This work

a See Materials and Methods for a description of specic strain constructions. Strains AM107 to STL1336 (lines 1 to 9 in the table) were used as donors for P1vir-mediated transduction. Strains MS100 to RM109 (lines 10 to 24 in the table) and RM125 (line 42) are derivatives of strain RW118 [thr-1 araD139 (gpt-proA)62 lacY1 tsx-33 glnV44(AS) galK2(Oc) hisG4(Oc) rpsL31 xylA5 mtl-1 argE3(Oc) thi-1 sulA211]. Strains RM110 to RM136 (lines 26 to 53 in the table), except for RM125, are derivatives of strain RW540 [line 25 in the table; thr-1 araD139 (gpt-proA)62 lacY1 tsx-33 glnV44(AS) galK2(Oc) hisG4(Oc) rpsL31 xylA5 mtl-1 argE3(Oc) thi-1 sulA211 leuB6(Am) uvrA6 recA730]. b The dnaN159 allele, which encodes two amino acid substitutions (G66E and G174A), was originally referred to as dnaN59 but has since been renamed by the E. coli Genetic Stock Center (CGSC). c These strains contain an unknown mutation that suppresses recA730 dnaN159 synthetic lethality. These suppressor loci are referred to as srd-1 and srd-2, for suppressor of recA730 dnaN159 synthetic lethality. See the footnotes to Table 3 and the text for further details.

Institute Biopolymer Facility, Buffalo, NY), fragments were subcloned into pWSK29 by digestion with EcoRI. The correct orientation (downstream of the lac promoter) was conrmed by diagnostic PCR using the M13 reverse primer, which is homologous to the sequence upstream of the multiple cloning site in pWSK29, paired with second primer homologous to the 3 -end of the cloned insert.

Transformation assays were preformed with the indicated E. coli strains which were made chemically component using rubidium chloride (51). Fifty microliters of component cells ( 5.2 108 cells) was incubated with 200 ng of each puried plasmid DNA on ice for 30 min. Reactions were heat shocked at 42C for 2 min, followed by incubation at 30C for 1 h. Aliquots of each reaction were then

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MAUL AND SUTTON TABLE 2. Plating efciencies of different isogenic lexA51(Def) strains

J. BACTERIOL.

Straina dnaN lexA

Relevant genotypeb polB dinB umuDC

Efciency of growthc (relative to growth at 30C): 35C 37C 39C

MS100 MS101 MS104 MS105 RM104 RM105 RM106 RM107 RM108 RM109
a b

159 159 159 159 159 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def)

1.1 1.4 1.0 3.0 10 1.2 1.0 10 4.3 2.6 2.1 4.0 10

1.1 1.1 1.4 2.0 10 1.4 2.0 10 3.9 1.9 1.1 8.1 10

1.9
3

1.0 10 4 0.41 1.0 10 6 0.75 1.0 10 6 3.2 1.0 10 6 2.9 1.0 10 6

Strains are described in Table 1. Abbreviations: , wild type; 159, dnaN159; 51(Def), lexA51(Def); , deletion; polB (Pol II), (araD-polB):: ; dinB (Pol IV), (dinB-yafN)::kan; and umuDC (Pol V), umuDC595::cat. c Representative clones for each strain were grown overnight in LB medium at 30C, and serial dilutions of each culture were plated at 30, 35, 37, and 39C. Values shown are the ratio of CFU observed at the indicated temperature after overnight incubation divided by the number observed for the same strain at 30C. The number of CFU/ml observed at 30C for each strain was: MS100, 5.3 108; MS101, 3.8 108; MS104, 1.9 108; MS105, 2.1 108; RM104, 2.4 108; RM105, 3.4 108; RM106, 0.73 108; RM107, 0.54 108; RM108, 0.65 108; and RM109, 0.59 108.

plated onto LB plates supplemented with ampicillin, and colonies were counted after overnight incubation at 30C. Mutagenesis assays. UV-induced mutagenesis was preformed using cultures grown overnight in M9 minimal medium supplemented with glucose (0.2%), thiamine (1 g/ml), and Casamino Acids (0.5%) essentially as described previously (56, 57). Briey, overnight cultures were subcultured in the same medium at 30C with shaking until they reached mid-exponential growth (optical density at 595 nm [OD595] of 0.6). One milliliter of culture was then transferred to a sterile glass 15 mm petri dish and either irradiated with 3 J/m2 UV light (254 nm) using a 15-watt germicidal bulb (General Electric) or mock-irradiated. Cultures were then transferred to sterile 25 mm glass bubbler tubes containing 9 ml of supplemented M9 medium, followed by overnight incubation at 30C with shaking. The following day, 100- l aliquots of the irradiated and mock-irradiated overnight cultures were plated in duplicate onto LB plates containing rifampin in order to measure mutagenesis, while appropriate dilutions were plated onto LB without rifampin in order to determine cell titers. UV-induced mutation frequency was then calculated by determining the number of rifampin-resistant (Rifr) colonies following UV irradiation minus the number of Rifr colonies induced by mock UV irradiation divided by the cell titer. Spontaneous mutagenesis was measured in a similar manner except that cultures were not UV irradiated.

RESULTS Access to the replication fork of the dinB-encoded Pol IV is regulated in part by its steady-state level. We previously reported that the lexA51(Def) allele exacerbated the temperature-sensitive growth phenotype of the dnaN159 strain such that it was unable to grow at temperatures above 34C (56). Since (i) the UV sensitivity of the dnaN159 strain was suppressed by inactivation of Pol IV and (ii) the dinB-encoded Pol IV is SOS regulated, we hypothesized that the enhanced temperature sensitivity of the dnaN159 lexA51(Def) strain was attributable to elevated levels of Pol IV. We tested this hypothesis in two different ways. In our rst experiment, we asked whether SOS-induced levels of Pol IV or one of the other SOS-regulated DNA polymerases (i.e., Pol II or Pol V) led to the growth defect of the dnaN159 lexA51(Def) strain. For this, we compared the growth properties of various isogenic lexA51(Def) strains bearing either dnaN or dnaN159 together with deletions of polB (Pol II), dinB (Pol IV), or umuDC (Pol V). Our results indicated that inactivation of Pol IV [ (dinB-yafN)::kan] suppressed the temperature-sensitive growth phenotype of the dnaN159 lexA51(Def) strain at both

35 and 37C (Table 2). In contrast, inactivation of Pol II [ (araD-polB):: ] or Pol V ( umuDC595::cat) had only a minimal effect on the growth phenotype of the dnaN159 lexA51(Def) strain. In our second experiment, we asked whether plasmids expressing DNA polymerase I, II, IV, or V impaired growth of the dnaN lexA(Def) and/or the dnaN159 lexA51(Def) strains described above. As shown in Fig. 1A, each of these plasmids transformed strain MS104 [dnaN lexA51(Def)] with an efciency that was comparable to that of the pWSK29 control plasmid. In contrast, we were unable to obtain more than a few transformants of the dnaN159 lexA51(Def) strain (MS105) using the Pol IV-expressing plasmid. With the exception of the Pol I-expressing plasmid, which displayed a 10-fold reduction in transformation efciency, the other plasmids behaved like the pWSK29 control plasmid with respect to their transformation efciency (Fig. 1A). Addition of isopropylthiogalactopyranoside (IPTG) to 50 M, which leads to maximal induction of the lac promoter in pWSK29 (57) (data not shown), had no effect on these results (data not shown). Very similar results were observed using strains RM110 [dnaN lexA51(Def) uvrA6] and RM111 [dnaN159 lexA51(Def) uvrA6] (Fig. 1B), indicating that, unlike the UV sensitivity of the dnaN159 strain that was severely enhanced by the uvrB::cat or uvrC:: cat allele (56), the Pol IV-dependent growth defect of the dnaN159 strain was independent of nucleotide excision repair. Taken together, these results suggest that LexA-dependent SOS regulation of the Pol IV steady-state level is important for helping to regulate its access to the replication fork. Synthetic lethality of the dnaN159 recA730 strain is suppressed by mutations that inactivate DNA Pol II or DNA Pol V. Results discussed above suggest that access of Pol IV to the replication fork can be regulated, at least in part, by its steadystate level relative to the other polymerases. RecA protein is required for induction of the global SOS response, as well as most homologous recombination, the repair of doublestranded DNA breaks, the restart of stalled replication forks, and Pol V-dependent translesion DNA synthesis (reviewed in references 11, 16, 24, 30, and 61). In order to determine whether RecA affects polymerase selection following SOS in-

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FIG. 1. Effects of plasmids directing expression of DNA polymerase I, II, IV, or V on growth of different dnaN and dnaN159 lexA51(Def) strains. Plasmids expressing Pol I, II, IV, or V were transformed into E. coli strains MS104 [dnaN lexA51(Def)] and MS105 [dnaN159 lexA51(Def)] (A) and RM110 [dnaN lexA51(Def) uvrA6] and RM111 [dnaN159 lexA51(Def) uvrA6] (B). Transformation efciencies are expressed as the number of CFU obtained per microgram of supercoiled plasmid DNA (note that the y-axis is a log10 scale). Values represent the averages and ranges of two independent experiments. Plasmids: pWSK29, control; pRM100, polA (Pol I); pRM101, polB (Pol II); pRM102, dinB (Pol IV); pRM103, umuD C (Pol V).

duction, we combined dnaN159 with different recA alleles under the premise that certain combinations might confer synthetic phenotypes that could be modied by mutations affecting one or more of the various E. coli polymerases. Inactivation of recA ( recA::cat) had only a minor effect on the dnaN159 strain, resulting in a slightly enhanced temperaturesensitive growth phenotype (data not shown), consistent with previous reports (19). This phenotype was similar to that of the dnaN159 lexA3(Ind ) strain (56), suggesting that it resulted from a lack of Pol V, and was not further characterized. We next examined recA alleles that displayed enhanced DNA binding activity. The recA441 allele, which bears E38K and I298V substitutions, displays an enhanced rate of association with ssDNA in vitro (25, 26). The recA730 allele, which was isolated from recA441 by Witkin et al. (70), contains only the E38K substitution, and the mutant RecA730 protein displays an even more robust ssDNA binding activity in vitro than does RecA441 (25). Despite the fact that we could efciently transduce the dnaN159 allele into the recA441 lexA51(Def) strain (RM112) by selection for kanamycin resistance conferred by the nearby zid-3162::Tn10kan allele (Table 3), transduction of dnaN159 into the isogenic recA730 lexA(Def) strain RW576 was remarkably inefcient (Table 3). Similar results were observed when trying to transduce dnaN159 into another recA730 strain [NR9350 (genotype: ara thi (pro-lac) sulA211 srlC300::Tn10 recA730) (13)] using a P1vir lysate prepared on a zid-3162::Tn10kan dnaN159 strain (data not shown), indicating that the inability to do so was specic to combination of dnaN159 and recA730 and not to some other allele(s). In order to rule out the possibility that the recA730 mutation suppressed the temperature-sensitive growth phenotype of the dnaN159 strain, we determined the nucleotide sequence of the PCR-amplied dnaN allele from six separate strains; all six corresponded to the wild-type dnaN sequence (data not shown). This result indicates that recA730 does not suppress the temperature-sensitive growth phenotype of dnaN159 and suggests that the combination of the dnaN159 and recA730 alleles results in a synthetic lethal phenotype. Given our nding that SOS-induced levels of Pol IV exacerbated the temperature-sensitive growth phenotype of the dnaN159 mutant (Table 2), we hypothesized that SOS-induced levels of one or more of the three SOS-regulated DNA polymerases, in combination with RecA730, might result in lethality in the dnaN159 strain. We therefore constructed a series of isogenic RW576 derivatives in which we combined the recA730 allele with mutations inactivating each of the SOS-regulated polymerases (Pol II, Pol IV, and Pol V) and tested these strains for their ability to be transduced to temperature sensitivity with dnaN159. In striking contrast to our results discussed above indicating that inactivation of Pol IV suppressed the growth defect of the dnaN159 lexA51(Def) strain (see Table 2 and Fig. 1), inactivation of Pol IV [ (dinB-yafN)::kan] had no discernible effect on the transduction efciency of dnaN159 into the recA730 strain (Table 3). Conversely, inactivation of either Pol II [ (araD-polB):: ] or Pol V ( umuDC595::cat) allowed efcient transduction of dnaN159 into the recA730 strain (Table 3). Furthermore, inactivation of both polB and umuDC in the same strain (RM126) resulted in an even slightly higher efciency of transduction for dnaN159 (Table 3). These ndings

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MAUL AND SUTTON TABLE 3. Genetic requirements for dnaN159 recA730 synthetic lethality
Relevant genotypeb Transduction efciency c Other No. of transductants selected (Tcr or Kmr CFU)

J. BACTERIOL.

Strain

recA

lexA

No. ts/total no. screened (% of total)

RW540 RM112 RW576 RM122 RM116 RM118 RM126d RM135e RM136e RM132
a b

441 730 730 730 730 730 730 730 730

51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def) 51(Def)

polB dinB umuDC polB, umuDC srd-1 srd-2 recR

94 89 552 1,227 37 65 616 920 2,008 58

40/48 (83) 41/50 (82) 2/94 (2)e 59/98 (60) 0/35 ( 3) 5/11 (46) 42/52 (81) 77/100 (77) 64/100 (64) 8/20 (40)

Strains are described in Table 1. Abbreviations: , wild type; 441, recA441; 730, recA730; 51(Def), lexA51(Def); polB (Pol II), (araD-polB):: ; dinB (Pol IV), (dinB-yafN)::kan; umuDC (Pol V), umuDC595::cat; srd-1 and srd-2, suppressors of recA730 dnaN159 synthetic lethality; and recR, recR252::Tn10 9kan. c Transductions were performed using a lysate propagated on a dnaN159 tnaA300::Tn10 strain except for strains RM112 and RM126, which used a lysate propagated on a dnaN159 zid-3162::Tn10kan strain (due to the fact that they contained the srlC300::Tn10 and fadR613::Tn10 alleles, respectively). The efciency of cotransduction of dnaN159 and tnaA300::Tn10 (or zid-3162::Tn10kan) was measured by patching out transductants in duplicate, followed by incubation overnight at 30 and 42C. Ratios represent the number of temperature-sensitive (ts) transductants divided by the number of clones patched out. Cotransduction frequencies for tnaA300::Tn10-dnaN159 (or zid-3162::Tn10kan-dnaN159) are indicated. Theoretical cotransduction frequencies were calculated to be 78% for tnaA300::Tn10 and dnaN, and 81% for zid-3162::Tn10kan and dnaN using the following formula: F (1 d/L)3, where F is the frequency of cotransduction, d is the distance between dnaN and zid or tnaA, and L is the size of the fragment that can be transduced by P1 vir (2.1 min) (40). d Transduction of the umuDC595::cat allele into recA730 (araD-polB):: strain RM122 was inefcient (data not shown), suggesting that Pol II or Pol V was required by (or at least important to) the recA730 strain. Therefore, we constructed a umuDC595::cat fadR613::Tn10 strain (RM125) so that we could measure the efciency of cotransduction of umuDC and fadR using the recA730 (araD-polB):: strain (RM122) and selection for Tcr. Cotransduction frequency for umuDC595::cat and fadR613::Tn10 was calculated to be 85%. Our nding that 39 of 45 (86%) Tcr transductants tested (from a total of 119 Tcr transductants) were also Cmr was in excellent agreement with the theoretical value of 85%, indicating that neither Pol II nor Pol V is required by the recA730 strain for viability. e The two tetracycline-resistant and temperature-sensitive dnaN159 recA730 strains identied were colony puried and named RM129 and RM130. The dnaN159 allele in strains RM129 and RM130 was replaced by dnaN using a P1vir lysate grown on the dnaN zid-3162::Tn10kan strain. Three hundred eighty-four Kmr transductants were obtained with RM129, while 408 were obtained with RM130. Thirty-nine of 40 tested for RM129 were temperature resistant, and 35 of the 39 temperature-resistant clones were Tcs. Thirty-eight of 40 tested for RM130 were temperature resistant, and 31 of the 38 temperature-resistant clones were Tcs. Four temperature-resistant and Tcs clones for each strain were picked and colony puried. One of each colony-puried strain was selected (named RM135 and RM136; see Table 1) for transduction with a P1vir lysate grown on the dnaN159 tnaA300::Tn10 strain, the results of which are shown above.

FIG. 2. Steady-state levels of the different umuD gene products in recA , recA441, and recA730 strains. E. coli strains were grown in LB medium to an OD595 of 0.5. A 2-ml aliquot of each culture was collected by centrifugation. Cells were resuspended in 20 l of sterile 0.8% NaCl and lysed by addition of 50 l of 4X sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading dye (200 mM Tris-HCl [pH 6.8], 8% SDS, 0.4% bromophenyl blue, 40% glycerol) containing 10% -mercaptoethanol prior to visualization by Western blot analysis using puried polyclonal antibodies specic to the UmuD and UmuD proteins. The salient features of each strains genotype are indicated. Strain examined: MS104, dnaN recA recR ; MS105, dnaN159 recA recR ; RM112, dnaN recA441 recR ; RM113, dnaN159 recA441 recR ; RM133, dnaN recA730 recR252::Tn10 9kan; RM134, dnaN159 recA730 recR252::Tn10 9kan; RM115, dnaN recA730 recR ; RM129, dnaN159 srd-1 recA730 recR ; and RM130, dnaN159 srd-2 recA730 recR . Levels of UmuD and UmuD in strains RM110 [dnaN lexA51(Def) uvrA6] and RM111 [dnaN159 lexA51(Def) uvrA6] were essentially identical to those observed in strains MS104 and MS105, respectively (data not shown).

indicate that lethality of the dnaN159 recA730 strain is the result of Pol II and/or Pol V. The fact that UmuD levels were similar in the recA441 and recA730 strains (Fig. 2) indicates that lethality is not due simply to the presence of elevated levels of Pol V or to the checkpoint function of umuDC (since intact UmuD was poorly detectable), but rather some property of the RecA730 protein in the dnaN159 strain which affects one or more functions of Pol II and Pol V. Genetic characterization of viable dnaN159 recA730 strains provides further evidence for a role of DNA polymerase Vdependent replication in conferring synthetic lethality. Despite the fact that the dnaN159 allele was synthetically lethal with recA730, we were able to identify two dnaN159 transductants of the recA730 strain that acquired temperature-sensitive growth (RM129 and RM130; see Tables 1 and 3). Based on nucleotide sequence analysis, these two strains did in fact contain the recA730 and dnaN159 alleles (data not shown). In addition, both strains contained the correct sequence for polB (Pol II) and umuDC (Pol V) (data not shown). In order to determine whether these strains contained a suppressor of the synthetic lethality, we rst replaced the dnaN159 allele in RM129 and RM130 with dnaN by selection for the nearby zid-3162:: Tn10kan allele (which is located on the opposite side of dnaN, as is tnaA300::Tn10), resulting in strains RM135 and RM136, respectively (see Table 3 footnotes). We next attempted to transduce strains RM135 and RM136 to temperature sensitivity with dnaN159 by selection for the nearby tnaA300::Tn10 allele. The results of this experiment indicated that both strains could be efciently transduced with dnaN159, demonstrating

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that each of these strains contained at least one mutation that did not map within polB or umuDC and was unlinked to dnaN that acted to suppress the lethal effects of dnaN159 in the recA730 strain (Table 3). The fact that strains RM129 and RM130 were viable but lacked mutations in polB and umuDC indicated that lethality could be suppressed by mutations affecting genes other than Pol II and Pol V. The RecF, RecO, and RecR proteins have been shown to play important roles in enhancing RecA function (37). We therefore asked whether the recR252::Tn10 9kan allele would allow efcient construction of a recA730 dnaN159 strain. As shown in Table 3, dnaN159 was transduced efciently into the recA730 recR252::Tn10 9kan strain. Nonetheless, nucleotide sequence analysis of the recF, recO, and recR genes from strains RM129 and RM130 revealed that each contained the correct sequence, indicating that suppression in these two strains was conferred by a mutation affecting a different gene(s). In order to better understand the basis for lethality of the recA730 dnaN159 strain, as well as the suppression of lethality by the recR252::Tn10 9kan, srd-1, and srd-2 alleles, we investigated whether these alleles exerted an effect upon Pol V-dependent UV-induced and/or spontaneous mutagenesis. It is known that RecA is required for catalytic activity of Pol V (48, 50, 62, 64). Thus, if recR252::Tn10 9kan, srd-1, and/or srd-2 impairs RecA function, Pol V function may also be impaired. Consistent with this hypothesis, the dnaN and dnaN159 recR252::Tn10 9kan strains displayed 5-fold and 2-fold lower UV-induced mutation rates, respectively, compared to the dnaN recR parent (Fig. 3A). Since these strains contained the lexA51(Def) allele, these results demonstrate an important role for the RecR protein in Pol V-dependent mutagenesis independent of SOS induction. Consistent with this nding, spontaneous mutation rates were modestly reduced in both the dnaN and dnaN159 recR252::Tn10 9kan strains (Fig. 3B). Thus, the phenotype of the dnaN159 recR252:: Tn10 9kan strain is consistent with a model in which deletion of recR suppresses synthetic lethality by impairing Pol V function. In order to determine whether srd-1 or srd-2 mediated suppression via an effect on Pol V function, we measured UVinduced SOS mutagenesis of strains RM129 and RM130. Pol V-dependent UV-induced mutagenesis was reduced 5-fold in strain RM129 (srd-1) relative to the dnaN recA730 control strain RM115 (Fig. 3A). In contrast, prociency in SOS mutagenesis of strain RM130 (srd-2) was similar to that observed for RM115 (Fig. 3A). We also measured spontaneous mutagenesis frequencies in both the dnaN159 recA730 srd-1 (RM129) and dnaN159 recA730 srd-2 (RM130) strains. While strains RM130 (srd-2) and RM115 displayed similar spontaneous mutation frequencies, spontaneous mutagenesis in strain RM129 (srd-1) was elevated more than sixfold relative to the dnaN control strain RM115 (Fig. 3B). Furthermore, this elevated mutation frequency was completely dependent upon Pol V, as the umuDC595::cat allele eliminated it (Fig. 3B). These results indicate that suppression of the dnaN159 recA730 synthetic lethality can be achieved simply by attenuating access to the replication fork of Pol V rather than affecting the catalytic activity of the enzyme per se. Based on these

FIG. 3. UV-induced and spontaneous mutation rates in different dnaN and dnaN159 lexA51(Def) recA730 strains. UV-induced (A) and spontaneous (B) mutagenesis frequencies were measured as described in Materials and Methods. Values presented are relative to the dnaN recA730 strain (RM115), which was set equal to 100%. Strain RM115 yielded 44.8 Rifr CFU/107 survivors for UV-induced mutagenesis and 2.2 Rifr CFU/107 survivors for spontaneous mutagenesis. Other strains examined: RM123, dnaN recA730 (araD-polB):: ; RM124, dnaN159 recA730 (araD-polB):: ; RM119, dnaN recA730 umuDC595::cat; RM120, dnaN159 recA730 umuDC595::cat; RM133, dnaN recA730 recR252::Tn10 9kan; RM134, dnaN159 recA730 recR252::Tn10 9kan; RM129, dnaN159 recA730 srd-1; RM130, dnaN159 recA730 srd-2; and RM131, dnaN159 recA730 srd-1 umuDC595::cat. Results shown are averages of at least three experiments standard deviation.

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FIG. 4. Growth rates of different dnaN and dnaN159 lexA51(Def) recA730 strains. Overnight cultures of E. coli strains grown in LB medium were subcultured into the same medium to a starting OD595 of 0.04. Cultures were grown at 30C with shaking, and the OD595 was measured and recorded every 20 min. Strains examined: RM115, dnaN recA730; RM123, dnaN recA730 (araD-polB):: ; RM124, dnaN159 recA730 (araD-polB):: ; RM119, dnaN recA730 umuDC595::cat; RM120, dnaN159 recA730 umuDC595::cat; RM133, dnaN recA730 recR252:: Tn10 9kan; RM134, dnaN159 recA730 recR252::Tn10 9kan; RM129, dnaN159 recA730 srd-1; RM130, dnaN159 recA730 srd-2; and RM131, dnaN159 recA730 srd-1 umuDC595::cat. Results shown represent the average of triplicates standard deviation.

results, we conclude that the srd-1 allele in strain RM129 affects the way in which Pol V is recruited to the replication fork. Functionally distinct roles for Pol II and Pol V in conferring recA730 dnaN159 synthetic lethality. In order to determine whether the dnaN159 recA730 synthetic lethality required the concerted actions of Pol II and Pol V or whether lethality resulted from the combined effects of their independent actions, we measured prociency in Pol V-dependent SOS mutagenesis of the dnaN and dnaN159 (araD-polB):: strains. As shown in Fig. 3, the frequencies of UV-induced and spontaneous mutagenesis in the dnaN and dnaN159 (araD-polB):: strains were within twofold of each other. Thus, taken together, these results indicate that Pol II does not signicantly affect Pol V function in translesion DNA synthesis, suggesting that synthetic lethality of the dnaN159 recA730 strain results from the combined effects of Pol II and Pol V and not from their concerted actions. To rule out the possibility that Pol II and Pol V were working together in some facet of replication that did not result in

an increased mutation frequency, but did confer lethality in the recA730 strain, we also examined the growth rates of the different recA730 strains. The rapid growth rate of the dnaN159 (araD-polB):: recA730 lexA51(Def) strain (RM124) suggests that inactivation of Pol II resulted in efcient suppression of synthetic lethality (Fig. 4). In contrast, the very slow growth rate of the dnaN159 umuDC595::cat recA730 lexA51(Def) strain (RM120) suggests that either (i) suppression of lethality by inactivation of Pol V is inefcient or (ii) despite its ability to confer lethality in the dnaN159 recA730 genetic background, Pol V nonetheless plays one or more important roles in the dnaN159 strain. This latter possibility was suggested by our previous observation that the umuDC gene products play an important role in protecting the dnaN159 mutant from the lethal effects of UV irradiation (56). In order to distinguish between these two possibilities, we examined the growth properties of the dnaN and dnaN159 recA730 lexA51(Def) strains bearing both the polB and the umuDC alleles. The results of this analysis indicated that

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inactivation of Pol II largely suppressed the poor growth of the dnaN159 umuDC595::cat recA730 lexA51(Def) strain (Fig. 4, inset). Thus, these ndings are consistent with a model in which Pol V competes with Pol II for access to the replication fork and that inactivation of Pol V alone suppresses synthetic lethality less efciently than does inactivation of Pol II. We also examined the growth rates of the recR, srd-1, and srd-2 strains in order to compare their efciency of suppression to those observed for the polB and umuDC strains. Our nding that strain RM134 (dnaN159 recA730 recR252:: Tn10 9kan) grew almost as well as the dnaN recA730 strain (Fig. 4) suggests that, in addition to impairing Pol V function, the recR252::Tn10 9kan allele also affects Pol II function, presumably by impairing RecA730 activity. Both the srd-1 (RM129) and srd-2 (RM130) strains grew as efciently as the strain carrying the (araD-polB):: allele, suggesting that these mutations were effective suppressors of synthetic lethality (Fig. 4). recA730 allele suppresses UV sensitivity of the dnaN159 strain. The model that begins to emerge from our results discussed above is that although transcriptional derepression of the SOS regulon appears to play an important role in allowing Pol IV to gain access to the replication fork, RecA protein subsequently facilitates access of Pol II and Pol V. If, in the course of promoting access of Pol II and Pol V to the fork, RecA mitigates access of Pol IV, either actively or as a result of increased competition with Pol II and Pol V, then it follows that the recA730 allele would suppress the UV sensitivity of the dnaN159 strain, much the same as the (dinB-yafN)::kan allele did (functional Pol IV, encoded by dinB, conferred UV sensitivity upon the dnaN159 strain [56]). As a test of this hypothesis, we measured the UV sensitivity of the dnaN159 recA730 srd-1 (RM129) and srd-2 (RM130) strains as well as the dnaN recA730 srd parent, RM115 (we were unable to examine the UV sensitivity of the dnaN159 recA730 srd strain due to the fact that it was inviable). As hypothesized, RM129 and RM130 were each indistinguishable from the dnaN parent strain with respect to UV sensitivity (Fig. 5). Although we do not yet know the nature of the srd-1 and srd-2 alleles, and hence we do not know the effect(s) that these mutations may have on UV sensitivity, these results are nonetheless consistent with a model in which RecA, either directly or indirectly, plays a role in attenuating access of Pol IV to the replication fork as part of the global SOS response. Consistent with this conclusion, Pol IV is reportedly inefcient at replicating a RecA-coated DNA template in vitro (32, 63, 67). DISCUSSION The steady-state levels and relative afnities of the various E. coli DNA polymerases for the clamp, as well as possibly other proteins that help to guide them to the replication fork, have presumably been optimized through evolution to ensure an ideal balance between accurate replication and capacity for translesion DNA synthesis. The goal of the work discussed in this report was to better understand the roles of LexA and RecA in mediating this process. We hypothesized that since Pol II, Pol IV, and Pol V are all SOS regulated, the global SOS

FIG. 5. UV sensitivity of E. coli srd-1 and srd-2 strains RM129 and RM130. Overnight cultures of strains RM115 (dnaN recA730), RM129 (dnaN159 recA730 srd-1), and RM130 (dnaN159 recA730 srd-2) grown in M9 minimal medium supplemented with glucose (0.2%), thiamine (1 g/ml), and Casamino Acids (0.5%) were subcultured into the same medium and grown at 30C with shaking until reaching an OD595 of 0.6. A 5-ml aliquot of each culture was irradiated as indicated previously (57), and appropriate dilutions of each irradiated and unirradiated sample were plated onto LB plates and incubated for two days at 30C. Results shown are the average of two independent experiments the range.

response might act to help manage the actions of these polymerases by regulating their access to the replication fork. As a test of this hypothesis, we used different lexA and recA alleles to determine the individual effects of SOS induction and RecA activation on Pol usage in the dnaN159 mutant. Our results indicate that inactivation of LexA (Table 2) or transformation with a plasmid directing expression of Pol IV (Fig. 1) is sufcient for this polymerase to impair growth of the dnaN159 strain. In contrast, both SOS induction and activated RecA protein, provided by the recA730 allele, were required in order for Pol II or Pol V to impair growth of the dnaN159 strain (Table 3). The fact that RecA730 is chronically active due to a mutation and therefore cannot return to a resting state appears to be vital for its synthetic lethality with dnaN159. Thus, our inability to observe a similar phenotype in a recA dnaN159 strain is presumably due to the fact that RecA is transiently activated in response to DNA damage and activated RecA levels dissipate as the damage is repaired or tolerated. Nonetheless, we have recently determined that the UV sensitivity of the dnaN159 umuDC strain is suppressed by inactivation of Pol II (M. D. Sutton and J. M. Duzen, submitted), indicating that Pol V and Pol II do confer a conditionally lethal phenotype in the recA dnaN159 strain following UV irradiation. Taken together, these results suggest that RecA protein, either directly or indirectly, inuences the ability of the different SOS-regulated polymerases to gain access to the replication fork in vivo (Fig. 6). Our nding that recA730 suppressed the UV sensitivity of the dnaN159 strain (Fig. 5) is consistent with a model in which

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FIG. 6. Model to describe the role of the E. coli sliding clamp, RecA protein and the global SOS response in managing polymerase usage at the replication fork. (A) Summary of the phenotypes observed for the dnaN159 mutant strain. Transcriptional de-repression of the SOS regulon by the lexA51(Def) allele leads to a conditional growth defect of the dnaN159 strain that is suppressed by inactivation of the dinB-encoded Pol IV. Subsequent introduction of the recA730 allele into the dnaN159 lexA51(Def) strain leads to a synthetic lethal phenotype that is suppressed by inactivation of the polB-encoded Pol II or the umuDC-encoded Pol V. (B) Model to describe the role of the SOS response in polymerase selection and switching in E. coli. Polymerases in bold are proposed to have the ability to gain access to the replication fork under the indicated conditions, and hence play prominent roles in DNA replication, while those in italics, although present, are proposed to play more minor roles. The lack of a polymerase indicates that it is not expressed to a signicant level (or is not active) under the indicated conditions. UmuD2C participates in DNA damage checkpoint control and is an inactive precursor to Pol V (38, 43). RecA/ssDNA-facilitated autodigestion of UmuD to yield UmuD serves to release the checkpoint while simultaneously activating Pol V (UmuD 2C) for translesion DNA synthesis. See the text for details.

RecA can impair access to the fork of Pol IV. However, it should be stressed that Pol IV can still presumably compete with Pol II and Pol V for access to the replication fork in the presence of activated RecA protein: indeed, the (dinB-yafN):: kan allele modestly enhanced Pol V-dependent UV-induced mutagenesis of a recA dnaN159 strain (data not shown), consistent with a model in which the RecA protein can act to attenuate competition between Pol IV and polymerases II and V. Thus, our ndings suggesting that the duration and the degree of SOS induction inuences polymerase selection (Fig. 6) could explain why the recA lexA holD::kan strain displayed a conditional lethality that was dependent upon Pol II, and to a lesser extent, upon Pol IV (65), while a similar dnaN159 strain displayed a conditional lethality dependent upon Pol IV (56): the holD::kan strain appears to be induced to a greater level for the SOS response than is the dnaN159 strain (56, 65), arguing that levels of activated RecA protein are higher. This, in turn, presumably results in the Pol II-dependent effect in the holD::kan strain. The inability of Pol V to impair growth of

the holD::kan strain may be due to the fact that activated RecA levels were insufcient to allow the accumulation of a sufcient steady-state level of Pol V to impair growth. Our nding that Pol II was more effective than Pol V at impairing growth of the dnaN159 recA730 strain (Fig. 4) is consistent with this model. Our nding that deletion of either polB (Pol II) or umuDC (Pol V) suppressed the lethality of the dnaN159 recA730 strain suggests that Pol II and Pol V are able to gain access to the replication fork in a RecA-mediated fashion. Our ndings that deletion of polB suppressed the growth defect of the dnaN159 recA730 umuDC595::cat strain (Fig. 4), and conferred a 2fold effect on Pol V-dependent mutagenesis in the recA730 strain (Fig. 3), irrespective of the dnaN allele, suggest that Pol II and Pol V compete with each other for access to the replication fork. Consistent with this conclusion, deletion of polB in the dnaN159 lexA51(Def) recA strain (RM105) resulted in a 17-fold increase in spontaneous argE3(Oc)3Arg reversion (data not shown). Subsequent deletion of umuDC conrmed that spontaneous mutagenesis was Pol V dependent, suggesting that in the dnaN159 strain, Pol II competed effectively with Pol V for access to the replication fork, effectively suppressing mutagenesis. The fact that Pol V requires RecA for catalytic activity indicates that RecA was activated in the cells that displayed Pol V-dependent spontaneous mutagenesis, consistent with our genetic analyses suggesting that RecA effects Pol selection. Given that 159 is impaired for interaction with the catalytic subunit of Pol III (56) and that lagging-strand Pol III must cycle to a new primer every 1 second during laggingstrand synthesis (39), it is possible that the conditional lethality of Pol IV in the dnaN159 lexA51(Def) strain is due to competition of Pol IV with the lagging-strand polymerase, impairing Okazaki fragment synthesis. Alternatively, Pol IV might compete with Pol I to impair Okazaki fragment maturation and/or ssDNA gap repair. Our observation that Pol I is essential in the dnaN159 strain (56) is consistent with the idea that this strain displays an increased dependence upon ssDNA gap repair. Hence, competition between Pol IV and Pol I and/or Pol III for access to nascent Okazaki fragments might result in persistent ssDNA gaps in the lagging strand. These gaps, in turn, could allow the formation of RecA/ssDNA nucleoprotein laments, leading to the chronic, low-level SOS induction observed in the dnaN159 strain (56). Pol II and Pol V might similarly compete with Pol III and Pol I for nascent Okazaki fragments, resulting in ssDNA gaps. However, our observation that the umuDC595::cat allele did not suppress the lethality of the dnaN159 polA::kan strain (data not shown) indicates that the polA::kan and recA730 alleles affect the dnaN159 strain in different ways. It was recently reported that dinB transcription is induced in E. coli by -lactam-mediated inhibition of cell wall synthesis in a lexA- and recA-independent manner (35, 44). Importantly, -lactam-mediated transcriptional induction of dinB correlates with an increase in the frequency of 1 lacZ frameshift mutagenesis in vivo (44). This nding is consistent with our results, suggesting that, in the dnaN159 strain, access to the replication fork of Pol IV is regulated largely by its expression level (although our results do not rule out the possibility that additional SOS-regulated gene products may be required for

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this process; see Table 1 and Fig. 1). The nding that Pol IV is expressed at 6 to 12 times higher steady-state levels than the other SOS-regulated polymerases is consistent with the idea that it is able to outcompete the other polymerases for binding to the clamp and subsequent access to the replication fork (23). This conclusion is further supported by reports that modest overexpression of Pol IV from a multicopy plasmid significantly increases the frequency of untargeted mutagenesis, indicating Pol IV-dependent replication (22). Thus, E. coli appears to utilize different control systems to manage the actions of each of its three SOS-regulated polymerases: the actions of Pol IV appear to be largely regulated by transcriptional controls, while the actions of Pol II and Pol V appear to be regulated in a far more complex manner: in the case of Pol V, these controls are incredibly complex and range from RecA/ssDNA-mediated autodigestion of UmuD to yield UmuD (7, 45, 53) to ClpXP- and Lon-mediated proteolysis of Pol V (14, 17, 18). In conclusion, our results indicate that the global SOS response plays important roles in helping to manage the actions of Pol II, Pol IV, and Pol V. Furthermore, our ndings suggest that RecA protein is able to attenuate the function of Pol II and Pol V as well as possibly Pol III and Pol IV in vivo. Further characterization of the roles of RecA protein in polymerase function as well as the identication of the srd-1 and srd-2 gene products will lead to a better understanding of polymerase management in E. coli.
ACKNOWLEDGMENTS We thank Mary Berlyn (E. coli Genetic Stock Center, Yale University) for advice regarding the nomenclature for the suppressor mutations in strains RM129 and RM130 and Roger Woodgate (NICHD, NIH) for his generous gift of antibodies specic to the UmuD/UmuD proteins. We also thank Mary Berlyn, Justin Courcelle (Portland State University), Benedicte Michel (Institut National de la Recherche Agronomique), Graham Walker (Massachusetts Institute of Technology), and Roger Woodgate for generously providing E. coli strains, Laurie Sanders for her comments on the manuscript, and the members of our laboratory for helpful discussions. This work was supported by Public Service Health grant GM066094 to M.D.S.
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