JOURNAL OF BACTERIOLOGY, Dec. 1990, p. 6885-6891 Vol. 172, No. 120021-9193/90/126885-07$02.00/0Copyright C 1990, American Society for Microbiology

Regulation of Photolyase in Escherichia coli K-12during Adenine Deprivation

JOSEPH L. ALCORNt* AND CLAUD S. RUPERTPrograms in Molecular and Cellular Biology, University of Texas

at Dallas, Richardson, Texas 75083

Received 29 May 1990/Accepted 24 September 1990

DNA photolyase, a DNA repair enzyme encoded by the phr gene of Escherichia coli, is normally regulatedat 10 to 20 active molecules per cell. In purA mutants deprived of adenine, this amount increased sixfold within2 h. Operon fusions placing lacZ under transcriptional control of phr promoters indicated no change intranscription rate during adenine deprivation, and gene fusions ofphr with lacZ showed a nearly constant levelof translation as well. Immunoblot analysis indicated that the total amount of photolyase protein remainedconstant during enzyme amplification. On the other hand, treatment of cells with chloramphenicol during theadenine deprivation prevented any increase. DNA regions lying 1.3 to 4.2 kb upstream of the phr codingsequences were necessary for this amplification to occur and for this purpose would function in trans. Theseresults suggest that adenine deprivation leads to a posttranslational change, involving synthesis of proteinencoded by sequences lying upstream of phr, which increases photolyase activity. The amplification in activitywas found to be reversible, for when adenine was restored, the photolyase activity declined before cell growthresumed.

Photoenzymatic repair of pyrimidine dimers induced inDNA by UV light is carried out by deoxyribodipyrimidinephotolyase (photolyase; EC 4.1.99.3) (31, 36). This enzymebinds to pyrimidine dimers and monomerizes them to con-stituent pyrimidines when the photolyase-dimer complexabsorbs a photon of wavelength in the range of 300 to 500 nm(11). In the absence of photoreactivating light, the boundphotolyase also plays an auxiliary role in endonuclease-mediated removal of dimers from DNA, the initial step ofexcision repair (28, 35, 45).The photolyase of Escherichia coli K-12 is encoded by the

phr gene located at 15.7 min on the linkage map (1, 15, 33).This gene has been cloned (34) and sequenced (39), and itsoverproduced product has been purified to homogeneity(40). The coding sequences slightly overlap an open readingframe (ORF169) upstream (39), which produces a polypep-tide of unknown function. The enzyme contains two chro-mophores: a neutral flavin radical that must be present in thereduced form for activity (20, 38) and a methenyltetrahydro-folate that is not essential for activity (18) but normallyabsorbs some of the photons driving the repair process (19).

Photolyase in E. coli is usually expressed at very lowlevels: 10 to 20 active molecules per cell in stationary phase(12) and only 1 to 2 per cell under anaerobic conditions (43).These small amounts severely limit photoenzymatic repair ofsolar UV damage during exposure to natural sunlight (8) andare suboptimal for the enzyme's auxiliary role in excisionrepair (45). The reason for regulation at such low levels is notunderstood. However, if cells carrying a purA mutation(phenotype Ade-) are deprived of adenine, the amount ofactive photolyase increases about sixfold (26, 34).

Earlier indications that the phr gene might be regulated bythe lexA gene product (16, 17) are not supported by subse-

* Corresponding author.t Present address: Department of Biochemistry, University of

Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,Dallas, TX 75235.

quent biochemical study (29), leaving the regulatory mech-anisms of phr unknown. In this work, we have investigatedthe level of control (transcriptional, translational, or post-translational) at which adenine starvation of purA mutantsproduces a photolyase increase. The results indicate that theamplification of active enzyme is posttranslational and that itrequires additional E. coli gene expression for its occur-rence.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains (all E. coli K-12derivatives) used in this study are listed in Table 1. The phrstrains were derived from MCL13 (M. C. Lorence, Ph.D.dissertation, University of Texas at Dallas, 1985) providingan isogenic background. JLA511 is a purA mutant in whichthe chromosomal phr gene is inactivated by insertion of thekanamycin resistance gene (Geneblock, Kanr gene; catalogno. 27-4897-01, Pharmacia P-L Biochemicals) at the StuI sitein the coding sequence. This strain was constructed byintegrating the altered phr gene from a plasmid, pCJL10-km(Fig. 1), using a method described by Winans et al. (44).GM48 was used to grow plasmids with nonmethylated DNA(25). P1 transductions were performed as described bySilhavy and Beckwith (41), using the bacteriophage P1Tn9clrl00.

Plasmid constructions. Restriction maps of relevant genesin plasmids pCJL10 (constructed by M. C. Lorence),pCSR606 (40), pMC1403 (6), pPCH1403 (6), pCJL11 (con-structed by M. C. Lorence), pCJL12 (constructed by M. C.Lorence), and pMS2 (39) and of the plasmids constructedduring this work are shown in Fig. 1. pCSR903 was con-structed by inserting the BclI fragment of pCJL11 into theBamHI site of pMC1403. pCSR906 was constructed byinserting the Stul fragment of pCSR903 into the unique StuIsite of pCSR606. Both pCSR903 and pCSR906 encode aphr::lacZ fusion protein, but pCSR906 has the complete 5'regulatory regions of pCSR606, while pCSR903 carries onlythe more restricted 5' sequences of pJCL11.

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TABLE 1. E. coli strains

Strain Relevant genotype Sourcea (reference)

ES4 purA45 CGSC 4431GM48 dam-3 dcm-6 CGSC 5127 (25)GR401 A(argF-lac)U169 CGSC 6710JC7623 recB21 recC22 CGSC 5188 (21)MCL13 A(kdpD-phr)214 M. C. LorenceJLA502 A(kdpD-phr)214 A(argF-Iac) MCL13 x P1/GR401

U169JLA505 A(kdpD-phr)214 A(argF-Iac) JLA502 x P1/ES4

U169 purA45JLA510 phr::kan JC7623 X pCJL10-kmbJLA511 phr::kan purA45 JLA505 x P1/JLA510

a Abbreviations: CGSC, Coli Genetic Stock Center, Yale UniversitySchool of Medicine (New Haven, Conn.); P1 transductions, recipient xPl/donor.

b JLA510 was constructed by transforming JC7623 with EcoRI-linearizedpCJL10-km and selecting for Kmr recombinants.

pCSR908 was constructed by inserting the SmaI-StuIfragment of pPCH1403 into the unique StuI site of pCSR606,resulting in a lacZ gene under transcriptional control of phrpromoters. pDHM1 (constructed by C.-P. Ma) is similar topCSR908, but insertion of the BamHI-MluI fragment ofpCSR606 into the BamHI site of pPCH1403 places lacZ 5bases after the phr termination codon but ahead of thetranscription terminator.

Chemicals and enzymes. Except when noted, all chemicalsand lysozyme were purchased from Sigma Chemical Co. (St.Louis, Mo.). Restriction endonucleases and T4 DNA ligasewere purchased from Bethesda Research Laboratories(Gaithersburg, Md.).

Culture media. Growth media were modified from thosedescribed by Maniatis et al. (24). KLB is LB with KCIsubstituted for NaCl. Minimal medium contained 11.3 g ofNaH2PO4 per liter, 7.0 g of KCI per liter, 3.0 g of K2HPO4

EI- t

B

B

E r

MblBct P tK i

Mb

A

St/S

BL

per liter, 2.0 g of NH4Cl per liter, 5.0 g of glucose per liter,1 mM MgSO4, 2 jig of biotin per ml, and 0.1 jig of thiamineper ml. Adenine (200 ,ug/ml) and ampicillin (125 jig/ml) wereadded where indicated.

Cell growth and adenine deprivation. Cultures were grownat 37°C with shaking. Cultures used for strain constructionand transformation were grown in KLB. Cultures used forassays were grown in minimal medium with adenine andampicillin.

1-Galactosidase assays. P-Galactosidase activity was de-termined by colorimetric assay as described by Pahel et al.(27).

Plasmid copy number. The plasmid copy number per celland per cell mass was determined as described by Lin-Chaoand Bremer (22), using densitometry.UV irradiation and photoreactivation. UV irradiation and

photoreactivation were performed as described by Harm etal. (12). Cultures were diluted in saline and magneticallystirred during irradiation. The UV source, a low-pressuremercury lamp (GE FG 445-8) emitting mostly 254-nm light,was controlled by a Compur Electronic 5 FS shutter. Cellswere irradiated at a fluence rate of 0.75 J/m2/s. Irradiationand subsequent manipulations were performed under yellow(nonphotoreactivating) light.

Photolyase activity determinations. Measuring the numberof active photolyase molecules in cells involves determiningthe number of pyrimidine dimers erased in single-flashillumination when the dimers are in excess of photolyase(12). This can be determined from the amount of UV fluencewhose effect has been nullified by the flash photoreactiva-tion, provided the amount ofDNA in the cells is known. Theamount of genomic DNA was determined by the Burtonassay (5) of known numbers of cells, while the amount ofUVfluence anulled in the single flash (the dose decrement [AD])was determined from the UV survival before and after flashillumination with the aid of the UV survival curve (9). The

j E pCJL1O-km

M0 E pCJL1O-J pCSR606.j pCJL11E0

.. pMS2

.i pCJL12;lacZ , StE pCSR908

phr M/B lacZ

Rr-IR nhrit**1 -7 Ct F

-t pDHM1

pCSR906Bc Bc/B phr::IacZ

St-ISB lacZ

SB bacZ'

St

pCSR903St

I pPCH1403St-I PMC1403

FIG. 1. Physical and genetic map of E. coli sequences in pCJL10 and its derivatives subcloned into pBR322 (3). Symbols: _, codingsequence from phr; -*, direction of transcription; L, lacZ coding sequences; *- , phr::lacZ fusion gene. The kanamycin resistance gene(Kmr) was inserted into phr at the position of the StuI site ( I ) to construct pCJL10-km. Restriction sites: E, EcoRI; E', EcoRI*; B, BamHI;Mb, MboI; A, AccI; P, PvuII; St, StuI; Bc, BclI; M, MluI; S, SmaI.

0 DU/ p . I cI =======a bmi

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PHOTOLYASE REGULATION DURING ADENINE DEPRIVATION

TABLE 2. Physiological effects of adenine deprivationin JLA505'

Time' Viable cells/ Genomes'/ Genomes/ Plasmids/ Plasmids/Tmime OD46 0D46 cell OD46 cell(m) (1O8) (108) (1010)0 3.9 8.4 2.2 2.4 6115 3.9 8.4 2.2 2.4 6130 3.9 8.4 2.2 2.4 6160 4.0 7.0 1.8 2.2 5590 5.2 8.6 1.7 2.0 39120 5.5 10.5 1.9 2.0 37

a Cells were grown in minimal glucose medium supplemented with adenine(200 ,ug/ml). Cells in exponential growth (OD4, = 0.2) were filtered from themedium and resuspended in the same volume of prewarmed adenine-freemedium.

b Measured from the start of adenine deprivation.Determined by Burton assay of culture DNA content (5), assuming 4.5 X

106 pg per E. coli genome.d Number of plasmids in JLA505(pCSR606), determined as described by

Lin-Chao and Bremer (22).

number of molecules of photolyase per cell, assumed equalto the number of dimers removed under these conditions,was calculated as (AD) x 65 x 0.85 x (genomes/cell), wherethe dose decrement is in Joules per square meter, 65 is thenumber of dimers produced per Joule per square meter perE. coli genome (32), 0.85 is the fraction of lesions induced byUV irradiation that is repairable by photolyase (11), and thenumber of genomes per cell is the number at the time ofirradiation and photoreactivation. The amount of photolyaseper unit of optical density at 460 nm (OD460) of cell culturewas calculated by multiplying the number of photolyasemolecules per cell by the number of viable cells per OD460 ofcell culture.Western immunoblot blot analysis. Indicated amounts of

bacterial culture were subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) (7). Transferand immunodetection were performed as described by Bur-nette (4). Monoclonal mouse antiphotolyase immunoglobulinG (M 22.5) was kindly provided by Aziz Sancar. Photolyase-antiphotolyase conjugate was detected by using 251I-proteinA (ICN).

RESULTS

Photolyase activity in adenine-deprived E. coli cells carryingphr on plasmids. The amplification of photolyase describedby Nishioka and Harm in adenine-starved purA mutants ofE. coli (26) involved the phr gene on the chromosome. Tosimplify genetic manipulations in studying its controls, weused purA A phr A lac E. coli strains with several multicopyplasmids incorporating the gene and flanking sequences.

Plasmid pCSR606 (Fig. 1) incorporates an E. coli DNAsegment beginning 4.3 kb upstream of the phr start codonand extending 77 bp past the termination codon. Whenexponentially growing cultures of JLA505(pCSR606) grownin minimal medium with adenine were rapidly filteredthrough a Millipore filter (0.45-pim pore size) and resus-pended in warm adenine-free medium, exponential growthcontinued at the same rate for another 45 to 60 min beforeabruptly creasing. The number of chromosome copies percell then underwent a small decline over the next hour (Table2).At various times after adenine deprivation, the cells were

irradiated and flash photoreactivated as described in Mate-rial and Methods. From the survival curves of the photore-

1l-

10-1'4

C4O"U:A_

E-

10

0 40 80 120 160 0 40 80 120 160

UV FLUENCE (J/m2)

FIG. 2. Colony-forming survival of JLA505(pCSR606) as a func-tion of UV fluence before and 2 h after adenine deprivation. Cellswere grown in minimal glucose medium with adenine, filtered duringearly exponential phase, resuspended in prewarmed medium with-out adenine, and irradiated as described in Materials and Methods.Closed symbols represent survival of cells with no photoreactiva-tion; open symbols represent survival following a 1-ms flash ofphotoreactivating light. The resulting AD values are indicated.

activated and nonphotoreactivated cultures, we were able todetermine the number of active photolyase molecules percell (Fig. 2). When the UV fluence is sufficiently high, theimprovement in survival produced by a 1-ms single-flashphotoreactivation is equivalent to decreasing the UV fluenceby an amount that is independent of fluence (the AD illus-trated in Fig. 2). The photolyase content is calculated fromAD and the physiological parameters listed in Table 2 asdescribed in Materials and Methods.The photolyase content per cell increased during 2 h of

adenine starvation (Fig. 3), as it did with phr on the chro-mosome (the number of enzyme molecules was, however,higher because of the higher gene copy number). Althoughthe number of plasmids per cell actually decreased (Table 2),the number of photolyase molecules per gene copy, whichbegan at about 12 and increased to approximately 113 duringstarvation, resembled that for the chromosomal gene of aK-12 strain under similar treatment, suggesting that theregulation is substantially the same.To determine the minimum amount of flanking DNA

required for regulation of photolyase, plasmids pCJL11,pCJL12, and pMS2, which carry phr with restricted 5'-flanking DNA, were tested for amplification of photolyaseactivity in the phr-deleted strain JLA505. Table 3 lists theamount of photolyase per cell before and after 2 h of adeninedeprivation. Unlike pCSR606, these plasmids showed littleor no amplification of photolyase activity, suggesting thatregions lying further than 1.3 kb upstream phr are necessaryfor regulation of photolyase.

Transcription ofphr during adenine deprivation. The com-plete 5'-flanking sequences from pCSR606, along with partof the phr gene, were placed upstream of a promoterlesslacZ gene (Fig. 1) to provide two operon fusion plasmids. Inplasmid pCSR908, only the 5' two-thirds of phr is present,giving no photolyase activity. Little change in the level ofP3-galactosidase activity in strain JLA505 harboring pCSR908occurred during adenine deprivation (Fig. 3), suggesting thateither the amplification is posttranscriptional or the operonfusion in some way interferes with normal transcriptionalregulation.

In pDHM1, the phr coding sequence is complete, but a

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0 30 60TIME (min)

90 120

*00 4

$ 3

0s :

I %1-1

QC

Before DeprivationAfter Deprivation

HIrp-galactouidame Photolyase

0so80

0x 70LJw 60

o 50(n0I- 40

i 30

%. 20U1.o 10O

-30 0 30 60 90 120TIME (min)

FIG. 4. Expression of 3-galactosidase and photolyase in JLA505(pDHM1) both before and 2 h after adenine deprivation. Bothactivities are plotted relative to cell mass (expressed as OD4w).

150

FIG. 3. (A) Photolyase content (active molecules per cell) ofJLA505(pCSR606), determined as described in Materials and Meth-ods. Open symbols represent cells deprived of adenine beginning attime 0; closed symbols represent control cells grown with adenineunder otherwise the same conditions. (B) Expression of ,3-galactosi-dase activity in JLA505(pCSR908), JLA505(pCSR903), and JLA505(pCSR906) during adenine deprivation. Cells were deprived ofadenine beginning at time 0. P-Galactosidase activity is plottedrelative to cell mass, expressed as OD4w.

promoterless lacZ gene is inserted downstream of it andupstream of the phr transcription terminator. UnlikepCSR908, pDHM1 expresses both active photolyase and3-galactosidase, since the two genes are transcribed from thesame phr promoters. The amount of active photolyase frompDHM1 was amplified sixfold during adenine deprivation, asin pCSR606, while the amount of 0-galactosidase decreasedslightly (Fig. 4). This result indicates that the transcription ofphr does not increase during the photolyase amplification.Also, photolyase must not autoregulate its own transcription

TABLE 3. Amount of photolyase in various strains

Active photolyaseStrain molecules/cell Ratioa

+Adeb -Adec

JLA505(pCSR606) 730 4,200 5.8JLA505(pCJL11) 1,220 1,780 1.5JLA505(pMS2) 610 730 1.2JLA505(pCJL12) 430 420 0.9JLA511(pCJL11) 1,700 7,140 4.2JLA511(pMS2) 970 3,250 3.4JLA505(pCSR606)d + 730 730 1.0

chloramphenicol

a Amount of photolyase molecules per cell after deprivation to amountbefore deprivation.

b At beginning of adenine deprivation.Two hours after adenine deprivation.

d Cells grown in the presence of 100 ,ug of chloroamphenicol per ml duringadenine deprivation.

during adenine deprivation, since the steady-state level ofphr::lacZ transcription, as measured by 0-galactosidase pro-duction, did not increase from that observed in pDHM1.

Translation of phr during adenine deprivation. PlasmidspCSR903 and pCSR906 both encode a fusion protein thatacts as P-galactosidase but not as photolyase. Production ofthese proteins depends on initiation of translation in phrsequences. The amount of P-galactosidase activity perOD460 of culture from these plasmids also did not increaseduring deprivation (although the number of active photol-yase molecules per OD460 was increased sevenfold by thistreatment, as calculated from Fig. 3 and Table 2), suggestingthat the amplification of photolyase is posttranslational innature (Fig. 3). The levels of P-galactosidase expression ofpCSR903 and pCSR906 were much less than that ofpCSR908 (Fig. 3); perhaps the initiation of translation ofphris less efficient than that of lacZ.Again the possibility of interference from the gene fusion

on normal phr regulation exists, as well as the possibility thatphotolyase itself is needed for the increase in its own

u,0

-j JLA505[pCSR6061Time 0 30 60 60A 90 120 120A

~ ~~~kDa92.-*2 .:.....

660-:"

Photolyase._..s450-

...,8z.* .. .. ....31> ::

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FIG. 5. Immunoblot analysis of photolyase production inJLA505(pCSR606) during adenine deprivation. For this assay, 0.5OD4w unit of culture that had been deprived of adenine for times upto 120 minutes was subjected to SDS-PAGE, electrophoreticallytransferred to nitrocellulose, and then immunoprobed with mouseantibody specific for E. coli DNA photolyase, followed by 125I1protein A. Numbers above the lanes indicate the time of growth (inminutes) without adenine, except for 60A and 120A, which indicate60 and 120 min, respectively, of growth with adenine.

4000

3000

2000

1000

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PHOTOLYASE REGULATION DURING ADENINE DEPRIVATION

800 -.--.Photolyses Molecules

0 - + + - Ad.,oz 700_

IX1OrmTo

10 5 1 0.5 0 120 120 Tinhe

600

Z 500

< 400-

300-

X 200 ade-D 100 - ade-.

> 0

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NUMBER OF PHOTOLYASE MOLECULES (X 1012)

FIG. 6. Photolyase protein content of JLA505(pCSR606) cellswith and without adenine deprivation, as determined by immunoblotanalysis. One OD4. unit of cultures treated as described and fourdifferent known amounts of purified photolyase protein were sub-jected to SDS-PAGE together, electrophoretically transferred tonitrocellulose, and then immunoprobed with mouse antiphotolyase,followed by 125I-protein A. The insert shows an autoradiogram ofthe resulting nitrocellulose sheet. Time is the time of growth (inminutes) with (+) or without (-) adenine. Densitometer scans ofthis radiogram give the plotted calibration curve as well as thereadings for cells deprived (ade-) and not deprived (ade+) ofadenine.

translation. We therefore performed immunoblot analysis ofphotolyase produced from pCSR606 before and after ade-nine deprivation. The amount of immunoreactive photolyasedid not change during adenine deprivation (Fig. 5), in agree-

ment with the conclusions from the results for gene fusions.These results suggest that only a fraction of the photolyaseprotein in the cells normally serves as active enzyme andthat this fraction increases during adenine deprivation.To determine whether the amplified amount of photolyase

after adenine deprivation represents all of the pool of pho-tolyase molecules or only a portion of them, strain JLA505harboring pCSR606 was grown in the presence or absence ofadenine for 2 h. Culture samples were then compared withknown amounts of pure photolyase (a gift of Aziz Sancar) byimmunoblot analysis, and the resulting autoradiograms ofthe photolyase bands were analyzed by densitometry (Fig.6). The results gave 2.2 x 1012 immunoreactive photolyasemolecules per OD460 before adenine deprivation and 2.3 x1012 molecules per OD460 after deprivation. From the data inFig. 3 and Table 2, we calculated 3.3 x 1011 active photol-yase molecules per OD460 before adenine deprivation, in-creasing to 2.3 x 1012 after deprivation in cells carryingpCSR606. Thus, the amount of active photolyase afteradenine deprivation appears to represent essentially all ofthe phr gene product in the cell.

Amplification is reversible upon addition of adenine. Returnof the increased photolyase activity in adenine-deprivedcells upon restoration of adenine presumably could occureither by inactivating active molecules or by diluting themout during growth and cell division (with low photolyaseproduction). When cells were deprived of adenine for 3 h andadenine was then restored, the number of active photolyasemolecules decreased within 30 min, before any increase inOD460 or viable cell count began sufficient to dilute them(Table 4), indicating fairly rapid inactivation of activatedmolecules.

Activation of photolyase by proteins encoded by upstream

TABLE 4. Photolyase content of adenine-deprivedJLA505(pCSR606) after adenine is restored to the medium

Timea Cells/OD4w Active molecules of Active molecules of(min) (101) photolyase/ODwb (1011) photolyase/cellb

0 5.0 23.0 4,60030 5.0 7.4 1,48060 3.6 3.8 1,050120 3.6 3.8 1,050a At beginning of adenine supplementation to the culture medium.b Determined as described in Materials and Methods.

regions of DNA. Evidently, the amplification of photolyaseduring adenine deprivation results from a posttranslationalmodification that increases the activity of photolyase mole-cules already present in the cell. However, addition ofchloramphenicol (100 ptg/ml) to the culture at the time ofadenine deprivation prevented the amplification (Table 3),indicating that some protein synthesis is necessary.The ancillary protein necessary for the amplification dur-

ing adenine deprivation might act in one of two ways. (i)protein produced during adenine deprivation could act withphotolyase (or synthesize a factor that acts with it), or (ii) theprotein produced might itself act as a secondary photolyaseduring adenine deprivation. Since the 4.2-kb DNA regionupstream of phr in pCSR606 must encode the necessaryprotein, we constructed JLA511, whose genomic phr gene isinactivated by insertion of the kanamycin resistance genewith the upstream region intact. This strain showed noevidence of photorepair, either before or after adeninedeprivation (data not shown), eliminating the second possi-bility.To determine whether the upstream DNA regions act in

trans to enable amplification of photolyase encoded onplasmids that lack this region, we tested pCJL11, pCJL12,and pMS2 in JLA511. These plasmids showed no amplifica-tion in JLA505 but showed photolyase amplification underadenine deprivation in JLA511 (Table 3).

DISCUSSIONThe sixfold increase in the number of active photolyase

molecules per E. coli cell when a purA mutant is deprived ofadenine stems from a posttranslational modification of inac-tive molecules. Only about 15% of the photolyase protein incells normally functions as active enzyme, while essentiallyall of it is active after adenine deprivation. This activation,which seems dependent on the translation product(s) ofDNA sequences 1.3 to 4.2 kb upstream ofphr, is reversed byadenine restoration.The activities of many enzyme molecules are modified in

response to metabolic needs. Mechanisms of activationinclude proteolytic cleavage, allosteric change of conforma-tion after binding an effector molecule, and covalent additionor removal of small molecular groups such as phosphate (13,14, 42). The latter two mechanisms would be directly revers-ible, while an activation by cleavage could be canceled by afurther molecular change. Photolyase is also known to beactivated by reduction and inactivated by oxidation of itsflavin cofactor (38, 39), another reversible alteration. Thereis little reason to favor one mechanism over another atpresent.

Highly purified photolyase in vitro shows an activitycomparable to that in the intact cell (36), with no otherprotein(s) essential for its full function. Consequently, it isunlikely that the activation involves complexing with some

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newly synthesized protein. Complexing with another proteincould inhibit the activity, which could then be restored byremoval. Less direct mechanisms might also be involved.The small number of active photolyase molecules usually

present in E. coli cells would seem reason enough foramplifying it under appropriate circumstances. At such a lowconcentration, its rate of binding to pyrimidine dimers (12)does not keep up with dimer formation in midday, temper-ate-zone sunlight, rendering photoenzymatic repair ineffec-tive under these natural conditions (10). Its only otherknown action, facilitating the UvrABC endonuclease's ex-cision of pyrimidine dimers to which it is bound, also seemssuboptimal, since the greater UV survival of recA strainswhich are phr' than of their phr mutant counterparts (45) isenhanced if the photolyase content is increased with amulticopy phr' plasmid (unpublished experiments). Sixfoldamplification of the photolyase would bring the rate ofenzyme binding more nearly in balance with the natural rateof damage formation, seemingly to the benefit of the cell.However, any connection between the cell's need for pho-tolyase activity and its starvation for adenine is a mystery;starvation of other E. coli auxotrophs for their essentialnutrient produces no similar amplification (26). Interestingly,the availability of ATP does regulate the biosynthesis of thephotolyase cofactors; GTP hydrolyase, which is the firstenzyme in flavin biosynthesis, is also the first enzyme infolate biosynthesis (2). However, none of the genes knownto be involved in biosynthesis of either cofactor are locatedin the vicinity of phr (1), eliminating any explanation for therequirement for protein synthesis in activation of photol-yase.The recA gene product, which is posttranslationally al-

tered in the presence of DNA damage to create the proteaseactivity initiating the cellular SOS response (23, 30), has asignificant role for genetic recombination without this alter-ation. The only functions known for photolyase concernrepair of pyrimidine dimers, and Phr- cells are at no evidentdisadvantage in the absence of UV radiation. However, thepossibility of other functions cannot be dismissed, and theapparent involvement with other gene products calls forclarification.

ACKNOWLEDGMENTSWe thank A. Sancar and M. Lorence for advice and criticism in

preparation of the manuscript.This work was supported by Public Health Service research grant

GM 16547 from the National Institutes of Health.

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