Vol. 139, No. 1JOURNAL OF BACTERIOLOGY, July 1979, p. 247-2550021-9193/79/07-0247/09$02.00/0

Pyrimidine Dimer Excision in a Bacillus subtilis Uvr- MutantCHARLES T. HADDEN

University of Tennessee-Oak Ridge Graduate School ofBiomedical Sciences, and Biology Division, OakRidge National Laboratory, Oak Ridge, Tennessee 37830

Received for publication 22 January 1979

A technique which allows the measurement of small numbers of pyrimidinedimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiatedwith ultraviolet light has been used to show that a strain mutant at the uvr-1locus is able to excise pyrimidine dimers. Excision repair in this strain was slow,but incision may not be rate limiting because single-strand breaks in DNAaccumulate under some conditions. Excision repair probably accounted for aliquid-holding recovery previously reported to occur in this strain. Recombina-tional exchange of pyrimidine dimers into newly replicated DNA was readilydetected in uvr-1 cells, but this exchange did not account for more than a minorfraction of the dimers removed from parental DNA. Excision repair in the uvr-1strain was inhibited by a drug which complexes DNA polymerase III with DNAgaps. This inhibition may be limited to a number of sites equal to the number ofDNA polymerase III molecules, and it is inferred that large gaps are produced byexcision of dimers. Because the uvr-1 mutation specifically interferes with excisionof dimers at incision sites, it is concluded that the uvr-1 gene product may be anexonuclease which is essential for efficient dimer excision.

Uvr- bacteria are characteristically very sen-sitive to UV irradiation. It was recently demon-strated that the survival of UV-irradiated uvr-1cells of Bacillus subtilis on nutrient agar platescould be enhanced if the cells were first incu-bated in a glucose-salts medium lacking aminoacids required for growth (16). For recovery inliquid medium, the cells had to be in stationaryphase and had to be mutant at the uvr-1 locus.This recovery also occurs if the cells are platedimmediately after irradiation on an agar mediumon which growth is slow. A speculative interpre-tation was that a defect in normal excision repairallowed detectable repair by a "different type ofexcision repair" (16), so that the cells were killedif growth resumed before about 3 h after irradia-tion.

Excision repair of damage to UV-irradiatedDNA often requires enzymatic removal of py-rimidine dimers (4, 22, 32). Endonucleases whichincise DNA containing pyrimidine dimers havebeen described (5, 8, 12, 34), but it is not clearwhich exonuclease completes the excision proc-ess. Escherichia coli uvr mutants are defectivein excision of dimers (4, 21, 30, 32), but to datenone of the known uvr mutants of E. coli hasbeen shown to have a defective exonucleasespecific for UV-irradiated DNA. Tests of strainsdeficient in exonucleases V, VI, or VII haveindicated that none of these enzymes is uniquelyrequired for dimer excision after low UV doses

(9, 11, 29), although a mutant lacking all threeexonucleases is severely limited in excision (25).Experiments presented in this and the accom-panying paper (18) imply that the uvr-1 locus inB. subtilis may code for an exonuclease requiredfor dimner excision. Dimer excision occurs in theuvr-1 strain, but, in contrast to Uvr+ cells, uvr-1cells of B. subtilis appear to leave large gaps atthe excision sites.

MATERIALS AND METHODSBacterial strains. The bacterial strains used were

GSY1026 (trpC2 metB4) (21), GSY1027 (trpC2 metB4uvr-1) (21), and FB56 (t7pC2 metB4 sfr uvs-42) (16).Growth of bacteria. For most experiments, cells

were grown at 37°C in a defined medium consisting ofmodified Spizizen salts (3), 0.5% glucose, 0.05% acid-hydrolyzed casein (Difco), and 50 Ag each of trypto-phan and methionine per ml. DNA was labeled with3H by inclusion of 30 ,ug of 2 '-deoxyadenosine and 20,uCi of [methyl-3H]thymidine (71 Ci/mmol, Schwarz/Mann) per ml.

Density-labeled cells were grown in a deuteriumoxide medium containing Spizizen salts (2), 5 mg ofdeuterated sugar mixture, and 50 ug of deuteratedamino acid mixture per ml (both from Merck and Co.,Inc.), 50 ,ug each of tryptophan and methionine per ml,and 1 MCi of ['4C]uridine (204 mCi/mmol, Schwarz/Mann) per ml. The cells were harvested by centrifu-gation, washed with modified Spizizen salts plus 0.5%glucose, and then incubated at 37°C at the originalconcentration in the (nonradioactive) light mediumdescribed above for 30 min to chase pools of heavy

247

248 HADDEN

isotopes. The cells were then harvested for irradiationas previously described (16).

Irradiation and postirradiation incubation.Cells were harvested by centrifugation, washed oncewith ice-cold modified Spizizen salts plus 0.5% glucose,and resuspended at a concentration of 5 x 107 to 7 x107/ml. They were irradiated with a 15 W germicidallamp, usually at a fluence rate of 0.375 J/m2 per s. Thecells were then diluted twofold into medium containingtwofold-concentrated supplements and incubated forthe indicated times. For inhibition ofDNA polymeraseIII, 6-(p-hydroxyphenylazo)-uracil (HPUra) was usedat 0.3 mM. HPUra was generously provided by B. L.Langley, Imperial Chemical Industries, Ltd., Maccles-field, England.

Spheroplast assay for pyrimidine dimers. Themethod for detecting pyrimidine dimers in sphero-plasts (C. T. Hadden, Microbios, in press) was modi-fied from Ganesan's procedure for E. coli (13). Con-ditions for alkaline sucrose gradient centrifugation andthe method for calculating molecular weights havebeen described previously (17).

RESULTSUV-induced incision and excision repair

in Uvr- strains. Most of the data below arepresented in terms of frequencies of incisionbreaks and ofdimers detected as sites for nickingby Micrococcus UV endonuclease (8, 33; R. B.Setlow, W. L. Carrier, and J. Stewart, Biophys.J. 15:194a, 1975) or as enzyme-sensitive sites(ESS). Figure 1 gives an example of the distri-bution of DNA from the wild-type strain

GSY1026 in sucrose gradients after irradiationwith 2.5 J ofUV per m2 and incubation in growthmedium for 30 or 60 min. The decrease in num-ber of ESS in DNA during postirradiation in-cubation shows that (i) dimers have been excisedand (ii) the excision sites have been fully re-paired. The breaks in endonuclease-treatedDNA result from (i) dimers remaining in theDNA at sites which have not been incised and(ii) sites at which incision has occurred but re-pair has not been completed. The latter alsoappear in samples which have not been treatedwith endonuclease. At these sites there may stillbe a pyrimidine dimer adjacent to (or very near)the incision site. By calculating from the molec-ular weight distributions the frequencies of sin-gle-strand breaks in the DNA (Table 1), it ispossible to estimate both the fraction of dimersremoved with complete repair and the totalnumber of incisions made in the process of re-pairing the DNA.The number of sites repaired is defined as the

reduction in number of breaks in UV endonucle-ase-treated DNA during postirradiation incuba-tion. This number may be an underestimatebecause it is assumed that the number of single-strand breaks made by the cell at sites otherthan pyrimidine dimers is negligible. That is,single-strand breaks at nondimer sites would becounted as unrepaired dimers. However, becausesteps in the excision-repair system are usually

15

io1NoEnzyme

LL

0 f70

0 10 20 0 10 20 0 10 20Bottom FRACTION NO. Top

FIG. 1. Alkaline sucrose gradient profiles of DNA from strain GSY1026. Stationary-phase cells labeledwith [3Hjthymidine were irradiated with 2.5 J of UVper m2 and incubated in growth medium at 37°C for theindicated times. They were then made into spheroplasts as described in the text, andpart ofeach sample wastreated with Micrococcus UV endonuclease. Conditions of centrifugation are described in the text. 0, No UVendonuclease; 0, plus UV endonuclease. (A) 0 min; (B) 30 min; (C) 60 min of incubation.

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DIMER EXCISION IN Uvr- B. SUBTILIS 249

TABLE 1. Number on incision and repair events in GSY1026a

Time (m n) UV fluence UV endo- (2/M,) x ESS Per i0 Sit repaired Unreired Total incisions(Jm) nuclease daltons incisions

0 0 - 10.12.5 + 46.3 36.2

30 2.5 - 11.7 1.62.5 + 20.2 8.5 26.1 27.7

60 2.5 - 11.8 1.72.5 + 15.8 4.0 30.5 32.2

a Data are from Fig. 1.

closely coordinated, the increase in number ofsingle-strand breaks in samples without endo-nuclease treatment remains small (Table 1), sothere cannot be many breaks at nondimer sites.These data are similar to those reported previ-ously by a number of authors (1, 17, 19). Incisionbreaks disappear by either completion of exci-sion repair or reversal of incision by ligase (30,31). Reversal by ligase cannot readily be de-tected, but completion of excision repair is de-tectable by the disappearance of ESS. The totalnumber of incisions can therefore be calculatedby adding the number of new single-strandbreaks to the number of sites completely re-paired. These results are then graphed as frac-tions of the initial dimer sites (Fig. 2).

Figure 2 shows the kinetics of total incisionand of completed repair in strains GSY1026(Uvr+), GSY1027 (uvr-1), and FB56 (uvs-42).Repair is rapid in the wild-type strain, and inci-sion is only in slight excess over complete repair.In strain FB56 there is hardly any completerepair, although incision breaks accumulateslowly. This confirms the lack of dimer excisionmeasured chromatographically (18). The uvr-1strain GSY1027 excises dimers slowly, but thereis only a slight accumulation of unrepaired inci-sion breaks. This implies in this strain eitherthat the incision step may be rate limiting, withessentially normal completion of repair once in-cision has occurred, or that incision may bereadily reversible, as appears to be true in E.coli uvrC strains (30, 31). This observation con-firms results from chromatography experimentsat higher UV doses (18).

Effect of growth conditions on repair inuvr-I celis. As noted previously (16), postirra-diation recovery of uvr-1 cells is prevented if thecells are incubated in nutrient broth or in thepresence of caffeine, or if the cells are harvestedfor irradiation during exponential growth. Figure3A shows that incubating irradiated stationary-phase uvr-1 cells in nutrient broth markedlydepresses repair of dimers. Incision is not re-duced, so incision and completion of repair seemto be at least partially uncoupled. A similar

100

(I)cnw-J

z

0 60 120TIME (min)

FIG. 2. Kinetics of incision and repair of ESS.Stationary-phase cells of strains GSY1026 (Uvr+),GSY1027 (uvr-1), and FB56 (uvs-42) were irradiatedwith 2.5 J of UV per m2 and incubated in growthmedium at 37°C for the indicated times before beingmade into spheroplasts and analyzed on alkalinesucrose gradients. Numbers of ESS repaired andtotal incisions were calculated as described in thetext and Table 1. 0, 4 O, Total incisions; 0, A, ,ESS repaired. 0, 0, GSY1026; A, A, GSY1027; O,E,FB56.

inhibition has been obtained by incubating uvr-1 cells in medium containing caffeine (C. T.Hadden and J. T. Lowry, Jr., unpublished data).Therefore, it appears that failure of recovery instationary-phase uvr-1 cells in nutrient broth isa result of inhibition of excision repair.

In contrast, incision and completion of repairare at least as rapid in exponentially growingcells as in stationary-phase cells in defined me-dium (Fig. 3B). Because exponentially growinguvr-1 cells die despite being able to removedimers from their DNA, it appears either thatslow excision of diimers cannot ensure survivalor that the dimers are being removed from pa-rental DNA strands by recombination into

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250 HADDEN100

(n

LJ

z

0 60 120 0 60 120TIME (min)

FIG. 3. Effect of growth conditions on repair ofESS in GSY1027. Exponentially growing or station-ary-phase cells of strain GSY1027 (uvr-1) were irra-diated with 2.5 Jof UVper m2 and incubated at 37°Cin either (A) Difco nutrient broth or (B) the aminoacid-supplemented glucose-salts medium. 0, A, E,

Total incisions; 0, A, , ESS repaired. Stationary-phase cells in (0, 0) defined medium or (A, A) nutri-ent broth; OZ, , log-phase cells in defined medium.

newly replicated strands.Exchange ofdimers into newly replicated

DNA. It was hypothesized previously (16) thatpostirradiation recovery involves recombina-tion-dependent excision of pyrimidine dimersbecause recovery was reduced in recA uvr-Imutants. Although it was subsequently shownthat the recA mutation affects metabolism ofrepaired DNA more than recombination (17),the possibility still remained that the disappear-ance of dimers was due solely to postreplicativerepair (27, 28) by exchange of dimers from pa-rental strands into newly replicated strands, as

has been shown in an E. coli uvrB mutant (14).However, the experiments described belowshowed that in stationary-phase uvr-1 cells,there was very little exchange of dimers, proba-bly because there was very little DNA replica-tion immediately after irradiation.Because the newly replicated DNA had no

radioactive label in the experiments describedabove, dimers exchanged into newly replicatedstrands would become undetectable withoutbeing removed from the cell. For recombinationof dimers into daughter DNA to be measured, itis necessary to know both the frequency of di-mers in the daughter DNA and the amount ofdaughter DNA. The frequency of dimers canbe determined by putting a radioactive label([3H]thymidine) in the postirradiation mediumand assaying ESS in the labeled DNA aftervarious times of incubation. The amount ofdaughter DNA can be estimated by growing thecells in a deuterium-based medium (with a 14Clabel as wel) before irradiation and in a light

medium afterward and determining by CsCldensity gradient centrifugation the fraction ofDNA replicated.

Figure 4 gives the sucrose gradient profiles ofDNA from strain GSY1027 in such an experi-ment. As in Fig. 1, the parental DNA withoutenzyme treatment was smaller after 60 min thanat 0 min, showing that some unrepaired incisionshad occurred. The samples treated with UVendonuclease show that some repair ofESS hadalso occurred by 60 min postirradiation. Figure4C shows that although some of the daughterstrands of DNA were very small, there was aconsiderable amount of daughter DNA that wasfairly large, with a few molecules as large as theunirradiated parental DNA. This larger DNAwas extensively nicked by UV endonuclease, somost of the large daughter DNA was associatedwith DNA which contained dimers. Becauseincubation in nonradioactive medium after ir-radiation but before labeling does not changethe sedimentation profiles (L. Dodson and C. T.Hadden, unpublished data), the large daughterstrands are not merely added to preexisting di-mer-containing DNA. Therefore, the dimersmust have come from the parental DNA byrecombination. Results are tabulated in Table 2.Figure 5 gives the kinetics of incision of parentalDNA, repair of parental DNA, exchange of di-mers into daughter DNA, and total excisionrepair, corrected for exchange of dimers out ofparental DNA. Although it is significant thatexchange of dimers into daughter DNA occurs,exchange does not account for the removal ofmore than a small fraction of dimers from pa-rental DNA; most dimers must be removed byexcision repair. More extensive exchange of di-mers occurs in exponentially growing cells, andin uvr-1 cells dimer excision takes place bothbefore and after recombinational exchange (L.Dodson, M.S. thesis, University of Tennessee,Knoxville, 1979).Formation of gaps at excision sites.

HPUra is a specific inhibitor of replicative DNAsynthesis (6, 7) which acts by inhibiting bothpolymerase and exonuclease activities of DNApolymerase III (24). In experiments in whichrepair synthesis was measured (18), it was as-sumed that DNA polymerase III plays, at most,an insignificant part in excision repair. Whenpolymerase III was inhibited by HPUra andrepair was measured by the UV endonucleaseassay, however, the surprising result was thatafter low UV doses incision occurred in strainGSY1027, but completion of repair was almostcompletely blocked by HPUra (Fig. 6B). Thiswas not true for the wild-type strain GSY1026(Fig. 6A), so it initially appeared that polymer-

J. BACTERIOL.

DIMER EXCISION IN Uvr- B. SUBTILIS 251

z0

cr-ILL

0 10 20 0 10 20 0 10 20Bottom FRACTION NO. Top

FIG. 4. Alkaline sucrose gradientprofiles ofparental and daughter DNA from strain GSY1027. Stationary-phase cells ofstrain GSY1027 (uvr-1) grown in deuterium oxide-based medium and labeled with ["4C]uridinewere irradiated with 2.5 J of UVper m2 and incubated at 37°C in light medium containing [3H]thymidine.Sam,ples were made into spheroplasts, and a portion of each sample was treated with UV endonuclease.0, No UV endonuclease; 0, plus UV endonuclease. (A) 0 min, parental [14C]DNA; (B) 60 min, parental[14C]DNA; (C) 60 min, daughter [3H]DNA.

100

n2JSit

0 60 120TIME (min)

FIG. 5. Repair and exchange of ESS in strainGSY1027. Data are taken from Fig. 4 and Table 2.0, Total incisions in parental DNA strands; 0, ESSremoved from parental DNA; A, ESS exchanged intonewly replicated DNA; A, net removal ofESS.

ase III was essential for the residual repair inuvr-1 cells. However, the relatively high levelsof repair synthesis and dimer excision (18) andrecovery from sensitivity to nutrient agar (16)by GSY1027 in the presence of HPUra were notcompatible with that conclusion.The mechanism of inhibition by HPUra is to

bind at the substrate site of polymerase III,making a tight complex comprising enzyme, in-hibitor, primer, and template (24). This is pos-sible only when polymerase III associates withgapped DNA. The inhibition ofrepair by HPUrais probably a result of the inhibited polymerase

III complex blocking the excision site, as shownby the following experiments with higher UVfluences. Because the number of polymerase IIImolecules is low in stationary-phase cells (per-haps on the order of 30 to 50 molecules per cell)(10, 15), it should be possible to titrate theenzyme with excision gaps in the presence ofHPUra. Additional gaps would then be free tobe repaired. This was indeed the case whenrepair in the presence of HPUra was comparedafter fluences of 2.5 and 7.5 J/m2. Figure 7 showsthat after 3 h of incubation of cells irradiated bya fluence of 2.5 J/m2 almost no repair had oc-curred in the presence of HPUra, although therewere about 10 unrepaired incisions per 109 dal-tons of DNA (or probably about 30/cell). After7.5 J/m2, there was a significant amount ofrepairdespite the drug, and again about 10 unrepairedincisions per 109 daltons of DNA. However, atboth fluences the total number of incisions wasonly about 1/3 as great in the presence of theinhibitor, implying that polymerase III may stillplay some role in the excision process.

DISCUSSIONBy use of an extremely sensitive enzymatic

assay to detect pyrimidine dimers, it has beenpossible to show that a Uvr- strain ofB. subtilis,GSY1027, can excise dimers after low UV flu-ences. In 2 to 3 h this strain can repair 60% ormore of the damage done by a UV fluence of 2.5J/m2. In contrast, another Uvr- strain, FB56

VOL. 139, 1979

252 HADDEN

(uvs-42), is unable to repair after even this lowfluence.The conditions for DNA repair in strain

GSY1027 appear to be the same as those forrecovery of ability to form colonies on nutrientagar (16), iinplying that the two processes arethe same. There is, however, one major excep-tion: although recovery of colony-forming abilitydoes not occur in log-phase cells, repair ofdimersdoes. Apparently by the time excision repair iscompleted under these conditions, some lethalevent, perhaps related to replication of dimer-containing DNA, has occurred.The nature of the uvr-1 lesion in strain

GSY1027 is not clear, but several possibilitiesmight be considered. (i) There might be a defectin UV endonuclease. This seems unlikely be-cause repair in the presence of HPUra or nutri-ent broth does not appear to be rate limited byincision. Furthermore, a defect in UV endonu-clease would not account for the apparent for-mation of large gaps at excision sites specificallyin the uvr-1 mutant (Fig. 6; reference 18). (ii)There might be a defect in excision by DNApolymerase I-associated exonuclease (11). Thisis unlikely because thepolA and uvr-1 mutationsmap in different regions (15, 20, 23). Unless theuvr-1 gene product is somehow required for ac-tivity of polymerase-associated exonuclease ashypothesized for E. coli recL (26), it is unlikelythat DNA polymerase I is responsible for asignificant portion of dimer excision in Uvr+ B.subtilis. The observation of Chase and Masker(9) that dimer excision is essentially normal afterlow fluences in poLA(Ex) mutants of E. colitends to support the view that the uvr-1 muta-tion is not like the polA(Ex) mutation. (iii)There might be a defect in a DNA binding orstructural protein. In E. coli uvrC mutants thereappears to be a defect in an activity whichnormally prevents DNA ligase from restoringincised strands before excision occurs, possiblyby binding to the incisedDNA (30) or by forminga complex which is essential for efficient dimerexcision (31). In a similar way, a defective DNA-binding protein or structural component of anendonuclease complex in B. subtilis GSY1027might prevent excision. Again, however, this ex-planation makes it difficult to account for therole of HPUra in blocking repair at low fluence.(iv) There might be a defect in UV exonuclease.This seems to be the simplest explanation forthe results presented above. My speculative in-terpretation is that the major portion (but notnecessarily all) of excision in Uvr+ cells after alow UV dose is done by a specific exonucleasecoded for by the uvr-1 gene. In strain GSY1027this enzyme is absent. Incision occurs but either

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J. BACTERIOL.

DIMER EXCISION IN Uvr- B. SUBTILIS 253

806

(fU)W 60-

z 40-0

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TIME (min)FIG. 6. Inhibition ofrepair ofESS in strain GSY1027 by HPUra. Stationary-phase cells ofstrain GSY1026

(Uvr+) and GSY1027 (uvr-1) were irradiated with 2.5J of UVper m2 and incubated at 37°C in growth mediumwith or without 0.3 mM HPUra. 0, A, Total incisions; 0, A, ESS repaired. O, 0, No HPUra; A,AP 0.3 mMHPUra. (A) GSY1026; (B) GSY1027.

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TIME (min)FIG. 7. Effect of UVfluence on inhibition ofrepair in strain GSY1027 by HPUra. Stationary-phase cells of

strain GSY1027 (uvr-1) were irradiated with 2.5 or 7.5 J of UVper m2 and incubated in growth medium withor without 0.3mM HPUra for the indicated times. 0, 4 Total incisions; 0, A, ESS repaired. 0, 0, 2.5 J/m2;A, A, 7.5 J/m2. (A) No HPUra; (B) 0.3mM HPUra.

is reversed by ligase or (infrequently) providessites by strand displacement or excision of di-mer-containing segments of DNA long enoughto allow DNA polymerase III to bind at theexcision sites. Inhibition of repair by the polym-erase III-HPUra complex indirectly implies theexistence of single-strand gaps in the DNA, be-

cause polymerase III is only inhibited by HPUrawhen it is associated with such a structure (24).This would explain the accumulation of single-strand breaks in the DNA during postirradiationincubation in the presence of HPUra.

Excision of dimners in oligonucleotides mightbe done by an endonuclease, or a single-strand-

VOL. 139, 1979

254 HADDEN

specific exonuclease might remove dimers bydegrading the displaced strand. It is clear thatthe excised fragment must be degraded at somepoint because dimers become acid soluble (18).The comparatively great inhibition of dimer ex-cision in the uvr-1 mutant is in marked contrastto the apparent ability of exonucleases to sub-stitute for one another in E. coli (9, 11). Thepossibility that the uvr-1 gene product is thesole enzyme used for normal dimer excision inUvr+ cells makes B. subtilis an attractive orga-nism for isolation of a specific UV exonuclease.

ACKNOWLEDGMENTSThis work was supported by U.S. Department of Energy

contract EY-76-S-05-4568 and the Office of Health and Envi-ronmental Research, U.S. Department of Energy, under con-

tract W-7405-eng-26 with the Union Carbide Corporation.I thank W. L. Carrier for the gift of Micrococcus UV

endonuclease and instructions for its use and B. L. Langleyfor the gift of the HPUra. I also gratefully acknowledge thetechnical assistance of A. S. Angel and G. R. Hellermann.

LTERATURE CITED

1. Achey, P., and D. Billen. 1969. Saturation of dark repairsynthesis: accumulation of strand breaks. Biophys. J. 9:647-653.

2. Anagnostopoulos, C., and J. Spizizen. 1961. Require-ments for transformation in Bacillus subtilis. J. Bac-teriol. 81:741-746.

3. Billen, D., L. B. Carreira, C. T. Hadden, and S. J.Silverstein. 1971. Evidence suggestive of compartmen-talization ofdeoxyribonucleic acid-synthesizing systemsin freeze-treated Bacillus subtilis. J. Bacteriol. 108:1250-1256.

4. Boyce, R. P., and P. Howard-Flanders. 1965. Releaseof ultraviolet light-induced thymine dimers from DNAin E. coli K-12. Proc. Natl. Acad. Sci. U.S.A. 51:293-300.

5. Braun, A., and L. Grossman. 1974. An endonucleasefrom Escherichia coli that acts preferentially on UV-irradiated DNA and is absent from the uvrA and uvrBmutants. Proc. Natl. Acad. Sci. U.S.A. 71:1838-1842.

6. Brown, N. C. 1970. 6-(p-Hydroxyphenylazo)-uracil: aselective inhibitor of host DNA replication in phage-infected Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A.67:1454-1461.

7. Brown, N. C. 1971. Inhibition of bacterial DNA replica-tion by 6-(p-hydroxyphenylazo)-uracil: different effecton repair and semiconservative synthesis in Bacillussubtilis. J. Mol. Biol. 59:1-16.

8. Carrier, W. L, and R. B. Setlow. 1970. Endonucleasefrom Micrococcus luteus which has activity towardultraviolet-irradiated deoxyribonucleic acid: purifica-tion and properties. J. Bacteriol. 102:178-186.

9. Chase, J. W., and W. E. Masker. 1977. Deoxyribonucleicacid repair in Escherichia coli mutants deficient in the5' 3' exonuclease activity of deoxyribonucleic acidpolymerase I and exonuclease VII. J. Bacteriol. 130:667-675.

10. Ciarrocchi, G., C. Attolini, F. Cobianchi, S. Riva, andA. Falasehi. 1977. Modulation ofdeoxyribonucleic acidpolymerase III level during the life cycle of Bacillussubtilis. J. Bacteriol. 131:776-783.

11. Cooper, P. 1977. Excision-repair in mutants of Esche-richia coli deficient in DNA polymerase I and/or itsassociated 5' 3' exonuclease. Mol. Gen. Genet. 150:1-12.

12. Friedberg, E. C., and J. J. King. 1971. Dark repair ofultraviolet-irradiated deoxyribonucleic acid by bacterio-phage T4: purification and characterization of a dimer-specific phage-induced endonuclease. J. Bacteriol. 106:500-507.

13. Ganesan, A. K. 1973. A method for detecting pyrimidinedimers in the DNA of bacteria irradiated with low dosesof ultraviolet light. Proc. Natl. Acad. Sci. U.S.A. 70:2753-2756.

14. Ganesan, A. K. 1974. Persistence of pyrimidine dimersduring post-replication repair in ultraviolet light-irra-diated Escherichia coli K12. J. Mol. Biol. 87:103-119.

15. Gass, K. B., and N. R. Cozzarelli. 1973. Further geneticand enzymological characterization of the three Bacil-lus subtilis deoxyribonucleic acid polymerases. J. Biol.Chem. 248:7688-7700.

16. Hadden, C. T. 1976. Postirradiation recovery dependenton the uvr-l locus in Bacillus subtilis. J. Bacteriol. 128:317-324.

17. Hadden, C. T. 1977. Repair and subsequent fragmenta-tion of deoxyribonucleic acid in ultraviolet-irradiatedBacillus subtilis recA. J. Bacteriol. 132:856-861.

18. Hadden, C. T. 1979. Gap-filling repair synthesis inducedby ultraviolet light in a Bacillus subtilis Uvr- mutant.J. Bacteriol. 139:239-246.

19. Harford, N., I. Samojlenko, and M. Mergeay. 1973.Isolation and characterization of recombination-defec-tive mutants of Bacillus subtilis, p. 241-268. In L. J.Archer (ed.), Bacterial transformation. Academic PressInc., London.

20. Hoch, J. A., and C. Anagnostopoulos. 1970. Chromo-somal location and properties of radiation sensitivitymutations in Bacillus subtilis. J. Bacteriol. 103:295-301.

21. Hoch, J. A., M. Barat, and C. Anagnostopoulos. 1967.Transformation and transduction in recombination-de-fective mutants of Bacillus subtilis. J. Bacteriol. 93:1925-1937.

22. Howard-Flanders, P., R. P. Boyce, and L. Theriot.1966. Three loci in Escherichia coli K-12 that controlthe excision of pyrimidine dimers and certain othermutagen products from DNA. Genetics 53:1119-1136.

23. Laipis, P. J., and A. T. Ganesan. 1972. A deoxyribonu-cleic acid polymerase I-deficient mutant of Bacillussubtilis. J. Biol. Chem. 247:5867-5871.

24. Low, R. L., S. A. Rashbaum, and N. R. Cozzarelli.1974. Mechanism of inhibition of Bacillus subtilis DNApolymerase Im by the arylhydrazinopyrimidine antimi-crobial agents. Proc. Natl. Acad. Sci. U.S.A. 71:2973-2977.

25. Masker, W. E., and J. W. Chase. 1978. Pyrimidinedimer excision in exonuclease deficient mutants ofEscherichia coli, p 261-265. In P. C. Hanawalt, E. C.Friedberg, and C. F. Fox (ed.), DNA repair mechanisms,ICN-UCLA Symposium on Molecular and Cellular Bi-ology, Vol. IX. Academic Press Inc., New York.

26. Rothman, R. H., and A. J. Clark. 1977. Defective exci-sion and postreplication repair of UV-damaged DNA ina recL mutant strain of E. coli K-12. Mol. Gen. Genet.165:267-277.

27. Rupp, W. D., and P. Howard-Flanders. 1968. Discon-tinuities in the DNA synthesized in an excision-defec-tive strain of Escherichia coli following ultraviolet ir-radiation. J. Mol. Biol. 31:291-304.

28. Rupp, W. D., C. E. Wilde, ml, D. L. Reno, and P.Howard-Flanders. 1971. Exchanges between DNAstrands in ultraviolet-irradiated Escherichia coli. J.Mol. Biol. 61:25-44.

29. Schlaes, D. M., J. A. Anderson, and S. D. Barbour.1972. Excision repair properties ofisogenic rec- mutantsof Escherichia coli K-12. J. Bacteriol. 111:723-730.

30. Seeberg, E., and W. D. Rupp. 1975. Effect of mutationsin lig andpoU on UV-induced strand cutting in a uvrC

J. BACTERIOL.

VOL. 139, 1979

strain of Escherichia coli, p. 439-442. In P. C. Hanawaltand R. B. Setlow (ed.), Molecular mechanisms for therepair of DNA. Plenum Publishing Corp., New York.

31. Seeberg, E., and P. Strike. 1976. Excision repair ofultraviolet-irradiated deoxyribonucleic acid in plasmo-lyzed cells of Escherichia coli. J. Bacteriol. 125:787-795.

32. Setlow, R. B., and W. L. Carrier. 1964. The disappear-

DIMER EXCISION IN Uvr- B. SUBTILIS 255

ance of thymine dimers from DNA: an error-correctingmechanism. Proc. Natl. Acad. Sci. U.S.A. 51:226-231.

33. Wilkins, R. J. 1973. Endonuclease-sensitive sites in theDNA of irradiated bacteria: a rapid and sensitive assay.Biochim. Biophys. Acta 312:33-37.

34. Yasuda, S., and M. Sekiguchi. 1970. T4 endonucleaseinvolved in repair of DNA. Proc. Natl. Acad. Sci. U.S.A. 67:1839-1845.