JOURNAL OF VIROLOGY, Dec. 1972, p. 1159-1169Copyright © 1972 American Society for Microbiology

Vol. 1O, No. 6Prtited in U.S.A.

Host- and Phage-Mediated Repair of RadiationDamage in Bacteriophage T4SUSAN S. WALLACE AND ROBERT J. MELAMEDE

Departmenit of Biological Sciences, Herbert H. Lehman College of the City Uniiversity of New York,Bronix, New York 10468

Received for publication 15 September 1972

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coliK12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensi-tivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4yalso show increased sensitivity on DNA polymerase-deficient strains, but to a

lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+,are plated on a double mutant lacking both DNA polymerase and the uvrA gene

product, a partial suppression of the polymerase effect is observed. Host ligase ap-

pears to be able to suppress to some extent the T4y phenotype but has no effect onwild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B,1 in the pres-

ence of chloramphenicol (50 jug/ml) shows increased resistance over directlyplated irradiated phage. Increased survival under the same conditions was notobserved with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was

investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30.The repair processes were further defined by observing the effects of plating ir-radiated phage on various hosts including strains lacking DNA polymerase I or

polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y

gene products seem to be involved in a repair system utilizing host ligase. Sec-ondly, in the absence of host DNA polymerase, phage sensitivity is increased in an

unknown manner which is enhanced by the presence of host ui rA gene product.

Although T4 and its Escherichia coli host bothcode for appropriate enzymes necessary to repairultraviolet damages to deoxyribonucleic acid(DNA), the relative role of these enzymes in re-pairing damage to the phage has not been fullyassessed. There are three genes which directly af-fect T4 ultraviolet sensitivity: v, x, and y (8, 18).The v+ gene codes for a dimer-specific endo-nuclease (13, 36), but the functions of x and yare unknown. It is suspected, however, that theyare involved in genetic recombination as well asrepair (7, 19). It has also been reported (2, 3)that temperature-sensitive T4 mutants defectivein DNA polymerase, ligase, nucleases, or gene 32protein are ultraviolet sensitive.

It has been generally assumed that host en-zymes play no role in the repair of ultraviolet-damaged T4 phage DNA, since survival curves ofT4 directly plated on repair-positive (B) and re-pair-negative (B,-1) hosts are identical (10, 17).Kozinski and Lorkiewicz (25) have shown, how-ever, that both of these hosts are capable of physi-cally nicking ultraviolet-irradiated T4, when phageenzyme synthesis is inhibited by chloramphenicol.This finding prompted us to investigate the effectof plating wild-type T4 and its ultraviolet-sensi-

tive mutants on a variety of repair-deficient hosts.In some experiments, phage-infected complexeswere held in chloramphenicol before plating, tobias in favor of host enzymatic repair.

It is clear that X-ray damages can also be re-paired since a variety of bacterial mutants existwhich have a wide range of sensitivities. Althoughthe nature of the X-ray lesions and their subse-quent repair is not well understood, there is apositive correlation between the ability to recom-bine and to survive ionizing radiation damage(21, 22). It also appears that recombinationalability is related to the repair of ionizing radiationdamage to T4 bacteriophages, since T4y and T4xwhich are ultraviolet and gamma-ray sensitivealso have impaired recombination ability (7, 19).It should be noted that there is a conflicting reportas to the sensitivity of T4x since Freifelder (12)has stated that this mutant is not X-ray sensitive.

Despite the potential usefulness of geneticallydefined bacteriophages for investigating the re-pair of X-ray damage, little has been done due tothe lack of sufficient mutants with altered survivalproperties. To further elucidate the possible re-pair mechanisms utilized by T4, temperature-sensitive mutants affected in the ligase or DNA

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WALLACE AND MELAMEDE

polymerase genes were examined for sensitivityto X rays. Both of these gene products have beenimplicated in the repair of ultraviolet damage byBaldy (2, 3), in the repair of ethyl methanesul-fonate (EMS) damage by Ray et al. (29), and ingenetic recombination by Bernstein (4). The X-raysensitivities of the above phage mutants, as wellas T4 wild-type, T4x, and T4), were also examinedin hosts lacking ligase or DNA polymerase I.A preliminary account of these studies has been

presented (S. Wallace and R. Melamede, Abstr.Biophys. Soc. 12:153a, 1972; R. Melamede andS. Wallace, Abstr. Biophys. Soc. 12:154a, 1972).

MATERIALS AND METHODS

Bacteria and bacteriophages. The properties of thebacterial and bacteriophage strains used in thesestudies are shown in Tables 1 and 2. The authors aregrateful to the indicated investigators for supplyingthe bacterial and phage mutants, and to H. Rosen-rkanz for giving us E. coli W31 10 and W3478.

Media. The bottom and top layer agar, broth, gelbuffer, and diluting fluid used were described previ-ously (33).

Ultraviolet irradiation. The bacteriophages were ir-radiated with a Sylvania 15-w germicidal lamp pow-ered through a Sola constant-voltage regulator. Phagesamples, in a 3.5-cm diameter petri dish, were irradi-ated with stirring under conditions of negligible ab-sorption. Radiation of wavelengths less than 250nm was removed by filtration through glacialacetic acid contained in a 7.5-cm diameter quartzcell (S. Wallace, Ph.D. thesis, Cornell University,Ithaca, N.Y., 1965). This procedure was utilized toeliminate wavelengths below 245 nm which damagethe protein coat of the phage (35). T4 wild-type phagesurvival curves were originally calibrated by usinguranyl oxalate actinometry (32). The D37 of T4+obtained with the filter was 80 ergs/mm'. Subse-quently, T4 wild-type bacteriophage was used as adosimeter, and the dose rate at 50 cm from the sourcewas 3.5 ergs per sec per mm2.X irradiation. The X-ray source was a Picker unit

with a beryllium window tube operated at 60 kvp

TABLE 1. Escherichia coli str-ains

Genotype withrespect to repair

WilduCvr-e vr-e

uvrA res*

sVr± res-1

uvrA res-1

Po/+polAl

lig+lop-8lop-8 lig-2

Phenotype with respect to repair

WildUV", X-ray sensitiveRadioresistantUV sensitive, slightly X-ray sensitive,

does not excise thymine dimersUV, X-ray sensitive, deficient in DNApolymerase

Double mutantWildUV, X-ray sensitive, deficient in DNApolymerase I

WildOverproduces ligase, UV sensitiveOverproduces defective ligase, UV

sensitive

Source

DoermannHillKondoKondo

Kondo

KondoCairnsCairns

GellertGellertGellert

References

20, 262323, 24

23, 24

23

8, 16, 28

151515

a UV, Ultraviolet.

TABLE 2. Bacteriophage T4 mnittanitsDesignation Phenotype with respect to repair Source References

T4+ Wild DoermannT4v UV, sensitive, deficient in a dimer excising en- Harm 13, 18, 36

donucleaseT4x UV, gamma-ray sensitive, recombination de- Harm 7, 19

ficientT4y UV, gamma-ray sensitive, recombination de- Boyle 7

ficientIT4tsB20 Temperature sensitive in gene 30-ligase, Edgar 2, 3, 11

slightly UV sensitiveT4tsL91 Temperature sensitive in gene 43-DNA po- Edgar 2, 3, 9

lymerase, slightly UV sensitive

a UV Ultraviolet.

Desi-nation

BB.,-,H/r30RHs30R

R15

OK2402W31 10W3478

N1071N1072N1 252

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REPAIR OF RADIATION DAMAGE IN PHAGE T4

and 20 ma. Phage samples in 9X citrate broth wereirradiated aerobically in 0.7-ml quantities with stir-ring. The dose rate was estimated by Fricke ferroussulfate dosimetry (34) and was found to be approxi-mately 70 krads, min.

Experimental procedure. Bacterial strains wereroutinely grown in broth at 37 C with aeration. Seedcultures were prepared from small inocula of freshovernight stationary-phase cultures. Thiamine hydro-chloride (0.2 mg/liter) was added to growth and plat-ing media for E. coli strains N1071, N1072, and N1252(15).

Lysates of T4x were prepared at 30 C as suggestedby Boyle (7). T4ts43 was prepared at 25 C, whereashigh-titer T4ts3O lysates were made at 30 C by usingE. coli strain N1072. Partially purified phage lysateswere diluted into citrate broth (9X) for X irradiation.For ultraviolet irradiation, phage lysates were par-tially purified by differential centrifugation and re-suspended in gel buffer.

Chloramphenicol complexes were prepared by in-fecting washed, log-phase cells with bacteriophage ata multiplicity of infecticn of 0.05 to 0.1. At the timeof infection, chloramphenicol was added to a final con-centration of 50 ug,'ml. If not otherwise indicated, thecomplexes were held for 30 min at 37 C with aerationand then plated and assayed for ultraviolet survival.Free phage were neutralized by anti-T4 serum 5 minafter infection. Anti-serum was prepared as describedby Adams (1), and the various preparations used hadK values ranging between 200 and 250 min-'. Phageadsorption was carried out in medium containing 20,ug of L-tryptophan per ml.

Survival curves were determined by diluting ir-radiated phage and plating on the indicated strains.Plates were usually incubated at 37 C. However,T4ts43 plates were incubated at 25 C and 43 C,whereas T4ts3O plates were incubated at 18 and 30 C.After ultraviolet irradiation, plating was done byusing dim yellow light to prevent photoreactivation.

All procedures not specifically described here havebeen published by Adams (1). The data presentedrepresent the average of at least three experiments,and, for the X-ray studies, generally five experiments.All the data were computer analyzed, and the finalslopes were determined from the best fit line obtainedby linear regression analysis. In all cases, the coriela-tion coefficient, r, fell within the 1% level of signifi-cance. In addition, for the X-ray data, standard devia-tions were determined for each experimental point.In no case, even for the data representing partial sup-pression (see Fig. 10), do the points overlap by onestandard deviation. Deviations are not presented onthe figures to facilitate visualization of the data.

RESULTSRepair of ultraviolet damages by host DNA

polymerase I. Figure 1 shows the survival ofT4+, T4x, T4y, and T4xv on pol+ and polAlcells. The latter strain is deficient in DNA polym-erase I (8) and is ultraviolet (16) and X-ray sensi-tive (28). T4+ and T4x exhibit increased sensi-tivity on polAJ, although T4x to a lesser extent

4."\ T4.

N.'

\*1 *\\

I .\~~~~~~~~~~~01~

0z

zv

-,

IA0 10 20 30 40 50

T4x

0,

0.1

1.0\I\ T4V

0.1

- 1.0

01

II\0.o1 z

0.01

ICT4xv

D S1UV DOSE SECONDS

FIG. 1. Ultraviolet suirvival of T4 mutawts oil pol+(0) anid polAI (E) hosts. A, T4+; B, T4x; C, T4v;D, T4xv.

than T4+. T4v and T4xv show no increasedsensitivity on the DNA polymerase-deficienthost. While these studies were in progress, Smithet al. (30) reported that wild-type T4 showed de-creased ultraviolet survival in a DNA polymer-ase-negative strain of E. coli and T4v did not.Apparently, the v gene product must be presentfor increased sensitivity to be observed in polAl.The survival of T4 temperature-sensitive gene

43 plated under permissive (25 C) or semi-re-strictive (43 C) conditions on pol+ and polAl isshown in Fig. 2. The same slight increase in sen-sitivity exhibited by this mutant on wild-type cellsis also observed in polAl. The increase in ultra-violet sensitivity exhibited by temperature-sensi-tive mutants on the T4 DNA polymerase gene(2, 3) has not been reproducible in our hands orthose of Speyer (31). Apparently, the reportedsensitivity increase is an artifact of plaque-count-ing technique (29). In any case, if T4 polymeraseplays a role in ultraviolet repair, it does not seemto be affected by the absence of host DNA polym-erase I.The ultraviolet sensitivities of T4+, T4y, T4x,

T4v, and T4xv on a series of suppressor-negative

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WALLACE AND MELAMEDE

C.,i

=

foC-

C.,

UV DOSE SECONDSFiG. 2. Ultraviolet survival of T4ts43 oni pol+, 25 C

(OI); pol+, 43 C (-); polAl, 25 C (0); anid polAl,43 C (0).

strains which lack DNA polymerase or ultra-violet-specific endonuclease function, or both,(22) are shown in Fig. 3. As can be seen, T4v andT4xv exhibit their mutant sensitivities inde-pendently of the strain they are plated on. T4 +shows the same increased sensitivity whetherplated on res-1, lacking DNA polymerase, orres-J uvrA, a double mutant which lacks bothDNA polymerase and ultraviolet-specific endo-nuclease function. Both of these strains are ultra-violet and X-ray sensitive (23, 24). No increasedsensitivity of T4+ or any of the mutants testedwas observed when plated on the uvrA mutant.The behavior of T4x in the res-1 strain mimicsthat observed when polAl served as host. Onres-1, T4y shows increased sensitivity, the rela-tive sensitivity being equal to that observed withT4x (Table 3). The similar response of T4x andT4y to the absence of host polymerase suggeststheir functioning in a common pathway distinctfrom that involving the v gene product and hostpolymerase. That T4x and T4y function in acommon pathway is in agreement with Boyle'sinterpretation of his radiobiological and geneticdata (5). Also, the sensitivity of T4x and T4y wasdecreased in res-l strains also lacking the uvrAgene product as shown in Fig. 3 and Table 3. Thisphenomenon was not observed with T4 +.

It should be noted that both polA1 and res-1

were derived by mutagenesis of their respectiveparent strains, and that the effect measured herecould be due to another mutation. We considerthis possibility unlikely, since both of these DNApolymerase-minus mutants exhibit identicalphenotypic responses with respect to repair.

Effect of host ligase on ultraviolet survival.Figure 4 shows the survival of T4 +, y, x, 1', andxv on lig+, lop-8 and lop-8 lig-2 bacterial hosts.The latter two strains overproduce active ordefective ligase, respectively (15). Only T4yshowed any ditference when plated on thesestrains, that being increased resistance in theligase overproducer. The response of T4 temper-ature-sensitive gene 30 in lop-8 and lop-8 lig-2 wasno different than when plated on wild-type bac-teria. The ultraviolet sensitivity of this mutantwas not exaggerated in a ligase-defective bacterialstrain.

Postirradiation incubation in chloramphenicol.Figure 5 shows the ultraviolet survival of T4mutants directly plated on strains B and B,_, ortreated at time of infection with 50 ,ug of chlor-amphenicol per ml and held at 37 C prior toplating. T4xv, but no other mutant tested, ex-hibited increased resistance on both B and B,1chloramphenicol complexes. The time course ofthis resistance increase is shown in Fig. 6. As canbe seen, the total increase appears to occur duringthe first 5 min of adsorption. Preliminary resultsindicate that T4xv exhibits the resistance increasein pol+ chloramphenicol complexes but not inpolAl chloramphenicol complexes. Thus, hostDNA polymerase and host nucleases are impli-cated in the resistance increase of T4xv in the ab-sence of phage-induced enzymes.

Phage-mediated repair of X-ray damages.Figure 7 shows the X-ray inactivation of tem-perature-sensitive T4 mutants in gene 43 (DNApolymerase) and gene 30 (ligase) at permissiveand nonpermissive temperatures. As can beseen, there is no increased X-ray sensitivity ofeither of these mutants at the nonpermissive

TABLE 3. Sensitivity comparison of T4 mnutanis onwild-type an)d miutanit bacterial straints

Final slope in mutant bacteria/final slopeBacteriophage in swil(l-type bacteria

mutant

T4+T4vT4xvT4xT4y

res-I

1.351.01.01.221.22

res-1 iur-A

1.351.01.01.081.11

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VOL. 10,1972 REPAIR OF RADIATION DAMAGE IN PHAGE T4 1163

T4+4Ty

0

a~~~~~~~~~~~~~~~~~

0.001

z~~ ~ ~~~~5~~~~~~~~~~~~

A

_ _ _ __6s 20m40 60 so

T4x TvT4xv

c

FIG. 3. Ultraviolet survival of T4 mutants on uvrA±res+ (0); uvrA-res+ (-); uvrA+res-1 (O); antd uvrA--res-l (v). A, T4+; B, T4y; C, T4x; D, T4v; E, T4xv.

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20 40 60 80 100 20

UV DOSE SECONDS

20 40 60 80 100

40 20 40

FIG. 4. Ultraviolet survival of T4 mutantts oni lig+ (0); lop-8 (H); and lop-8 lig-2 (A). A, T4+; B, T4y; C,T4x; D, T4v; E, T4xv.

temperature tested, indicating the apparent lackof involvement of these mutants in repair of X-raydamage. The X-ray sensitivity of T4x is 11 6%Omore sensitive than wild-type phage (Fig. 8). T4yon the suppressor-negative host also exhibits 11 %0increase in sensitivity (Fig. 8). The sensitivity ofT4x and T4y measured here with X rays is notas great as that reported by Boyle and Symonds(7) for gamma rays. It is theoretically possiblethat the gamma rays, which have a lower linearenergy transfer than X rays, could inflict a pre-

ponderance of a different class of reparable dam-ages than X rays.

Host-mediated repair of X-ray damages. Theeffect of host DNA polymerase I on the X-raysurvival of T4+, T4x, and T4ts43 at permissive

and nonpermissive temperatures is shown in Fig.9. All three phage strains tested showed increasedsensitivity on polAl to the same extent. The ab-sence of phage DNA polymerase did not enhancethe effect observed in the absence of host DNApolymerase I.

The X-ray sensitivities of T4+ and T4'on

suppressor-negative strains lacking DNA polym-erase or uvrA gene product are shown in Fig. 10.The results obtained on res-J with T4+ as wellas T4x (not shown) are similar to those observedwith polAl. Also, T4y exhibits increased sensi-tivity on DNA polymerase-negative res-]. Al-though there is no observable effect when T4 isplated on the uirA mutant, the polymerase-minus

T4y

ff0

B

0z

A

z0

ofM-

0.1

0 oi

T4x

S

A

C~~~~~~~~

\*~~~~~~~~~~~~c~~~

\2"~I T4v

D

T4xv

A

I

a

E

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REPAIR OF RADIATION DAMAGE IN PHAGE T4

1o 20 30 40 50UV DOSE S

1165

lU

iECON DS

FIG. 5. Ultraviolet survival of T4 mutatnts directly plated oni B (0) or B,-l (A) hosts; or plated after postir

radiationi inicubationi of B (0) or B,-l (A) phage complexes in chloramphenzicol. A, T4+; B, T4x; C, T4v; D

T4xv.

sensitivity of all phage strains tested was partiallysuppressed on the uvrA res-J double mutant.

Figure 11 shows the X-ray survival of T4 +and T4y on wild-type, lop-8, and lop-8 lig-2strains. The latter two hosts overproduce activeor defective ligase, respectively. Wild-type T4,but neither T4y, nor T4x (not shown), exhibitedincreased sensitivity in the strain overproducingdefective ligase.The survival of the temperature-sensitive gene

30 mutant when plated on lop-8 at permissiveand nonpermissive temperatures is identical tothat of the wild-type phage. However, when thegene 30 mutant was plated at nonpermissive tem-peratures on the lop-8 lig-2 host, it did not exhibitthe increased sensitivity of the wild-type phage. Alikely explanation for the difference in sensitivityis that the nonpermissive temperature for thephage is in fact permissive for the temperature-sensitive lig-2 mutation (27), thus rendering thehost,'in effect. lop-8.

DISCUSSION

Wild-type T4 showed a 35%, increase in ultra-violet sensitivity in DNA polymerase-deficientstrains of bacteria. This agrees closely with the30 observed by Smith et al. (30) without filter-ing the germicidal lamp output. The increasedsensitivity on pol minus hosts was not observedwith T4v or T4xv, indicating that the v+ geneproduct may be functioning with DNA polymer-ase I, presumably during excision repair. In theabsence of DNA polymerase I, nuclease actionobserved by Boyle et al. (6) in polAl cells mightdegrade at v gene product-induced nicks. Itshould be noted that the lack of bacterial polymer-ase affects v gene repair only to a minor extentsince the increase in slope is 35% compared toabout 120%C in the absence of the v gene function.

Although v+ gene product appears to be re-

sponsible for nicking during ultraviolet excisionrepair in T4, under special circumstances host

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0

z

z

0

I-

'I

a

.\2

o.l~~~~~~A

i

A

1.0~~~~~~~~~~

a T4 v

0.1

0.01 \

C\C

T4x

B

T4 xv

\ 0

A 0

0

D a

in 7n in In rnJu 4u Wu

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WALLACE AND MELAMEDE

observe biological repair. Similarly, host biologi-cal repair of wild-type T4, as well as single T4

2.5 * mutants, was not observed in these studies pre-sumably because phage repair systems mask theeffect in a manner analogous to the masking ofliquid holding recovery in certain strains of bac-teria (14).

CZ / T4x and T4y showed a 22' increase in ultra-_ 20 A/ violet sensitivity in DNA polymerase-deficientbacterial strains. This effect was partially sup-pressed in DNA polymerase-deficient strains alsolacking in the uvrA gene product. Suppression

1.5 /did not occur with wild-type T4. To account forthis observation, we propose that, in the absenceof x-y type repair, v type excision repair is ex-

.,-/̂ o_tended. During extended excision repair, en-______/_o_ dogenous uvrA product, v gene product, host-AOSOi0RP1ION-_ 0 i 1i- | DNA polymerase, and other enzymes involved inAOSORPTION--w-D 5

-0

excision repair could function to repair the dam-ADDITION MINUTES aged phage DNA. In the absence of host polymer-Iase, removal of the uvrA product would increaseFIG. 6. Time course of resistance inicrease of T4xv

in chlorampheniicol-treated complexes of B (0) ancdB,_1 (A); a,id in unitreated complexes of B (0) anid 1.0Bs,, (A). Data are also shownI for T4+ in chlor-ampheniicol-treated complexes of B (O) antd B,_, (*).

T4t,43 r03

0,1 0.1

0.01A ~~~~~~~~~z

z~ ~ ~ t >e lo- 5 -

0~~~~~~~~~~~~~~~~~~~~~~~~~~>~~~~~~~0h.01

0.001

z2

A~~~~X-RAY DOESEOD

FIG. 7. X-ray survival of temperaiturie-sentsitivemultants ont F. coli B. A, T4ts43, 25 C (E]) anid 43 C 0.001(U). B, T4ts3O, 18 C (0) and 30 C (0).

nucleases seem to be able to perform this func-tion. This is evidenced by increased ultravioletsurvival of T4xv in B and B1-1 when incubated in A

chloramphenicol, in the absence of phage-in- A

duced biosynthesis. Kozinski and Lorkiewicz ob- 50 100 150 200served physical nicking and repair of ultraviolet- X-RAY DOSE SECONDSirradiated wild-type T4 in chloramphenicol com- FIG. 8. X-ray survival oni E. coli B of T4+ (-)plexes of both B and B9,-; however, they did not T4x (A), and T4y (0).

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REPAIR OF RADIATION DAMAGE IN PHAGE T4 1167

SO 100 1SO SO 100 1SO SO 100 1SO

X-RAY DOSE SECONDS

FIG. 9. X-ray survival of T4 mutants on DNA polymerase-positive or -negative hosts. A, T4+ on pol+ (0)anid polAl (@). B, T4x on pol+ (A) and polAl (-). C, T4ts43 onf pol+ 25 C (0), pol+ 43 C (-); polAl25 C (@), polAI 43 C (O).

az

z

1I-W6

X-RAY DOSE SECONDS

FIG. 10. X-ray survival of T4+ (A) and T4y (B) on uvrA+res+ (0); uvrA-res+ (0); uvrA+res-l (L);

aiid uvrA-res-l (U).

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U

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0.001

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T4+

0.1

z

Z 0.010

0.001

X ~~~~~T4y

Is

'I

so 100 5SO 200 50 o00 I50 200

X-RAY DOSE SECONDS

FIG. 11. X-ray survival of T4 + (A) a,lit T4y (B)ont lig+ (@); lop-8 (U); anud lop-8 lig-2 (A).

resistance though not necessarily back to themutant phage phenotype. The implication is thatnuclease action present in DNA polymeraseminus hosts is making lethal damages out ofaivlrA nicks occurring at nonlethal ultravioletdamages or at nonspecific sites. Suppression of thepolymerase effect would not occur with wild-typephage because the x-Y pathway would be function-ing and excision repair would not be extended. Itshould be noted that Kato and Kondo (24) founddecreased breakdown of DNA after ultravioletirradiation of the uvrA res-I double mutant com-

pared to the res-] mutant alone. The response ofT4y to the ligase overproducer is difficult to ex-plain in the absence of further information.We are presently studying functional survival

by using T4 ultraviolet-sensitive mutants in a

variety of mutant bacterial strains. Hopefully, we

shall gain enough information from this type ofapproach to adequately assess the role of host en-

zymes in ultraviolet repair of T4 and to providefurther insights into the nature of the phage-codedrepair systems. Ultimately one should be able toprovide a point of departure for elucidating theenzymatic nature of the x and y gene products.The repair processes that occur following X

irradiation are not well defined. Without ap-propriate mutants possessing altered sensitivities,there has been little benefit in using the relativelysimple phage systems for studying X-ray repair.To investigate the possible involvement of specificgene products in X-ray repair, temperature-sensi-tive T4 mutants were examined for X-ray sensi-tivity under the same semirestrictive conditionswhich permit expression of ultraviolet sensitivity.The results indicate, at least to the limit of resolu-

tion of these measurements, that phage-codedDNA polymerase and ligase are not involved inthe X-ray repair process. However, these sameenzymes, when coded for by the host, appear toplay a role in determining phage survival, sincethe absence of either of these products results inan increased sensitivity to X rays. The sensitivityis equal to that observed by us when using theT4x or T4y mutants. By plating X-irradiatedT4x and T4y, both of which appear to be involvedin the same repair pathway (5, 29), on variousmutant hosts, it is possible to demonstrate twoapparently distinct processes that atTect phagesurvival. When both the x and y gene products arepresent, the lack of host ligase results in an 11 cincrease in phage sensitivity to X irradiation.When either of these phage gene products arelacking, there is no host-mediated sensitivity in-crease observed on the ligase minus host. It ap-pears, therefore, that host ligase might function inconjunction with the x and y gene products inrepairing X-ray-damaged phage DNA.

All phage strains tested showed increased sensi-tivity to X rays when plated on DNA polymerase-negative hosts. This is similar to the results ob-tained with ultraviolet-irradiated phage, with theexception of T4v, and for EMS-treated T4 (29).To evaluate the possible repair role of the hostDNA polymerase I, one can look at the partialsuppression of the polymerase minus sensitivityfound when the host is also mutant at the uIllAlocus. A possible explanation for the observedphage-host relationship involves the considera-tion of two types of single-stranded breaks: thosethat are directly caused by the X rays, and thosethat result from the uvrA gene product acting onsome X-ray-induced helical distortions whichcould possibly result from base or sugar damages.When only polymerase is lacking, both types ofdamages could become sites for lethal degrada-tion. When uvrA gene product and DNA polymer-ase are absent, only the single-stranded breaksthat are directly caused by the X rays are lethal.A further complication, that results when tryingto develop a working hypothesis regarding the re-pair of X-ray damage to T4, is the as yet unre-solved question of whether the lack of host DNApolymerase prevents the repair of lethal damagesor in fact allows for their creation. The increasedDNA degradation observed when DNA polym-erase minus bacteria are X irradiated (6), or whenT4 is X-irradiated and plated on the polymerase-negative host (Marsden and Ginoza, personalcommunication), appears to be evidence for thelatter phenomenon.When one takes into account both systems

which we have found to affect phage survivalafter X-irradiation, we can explain a 22%- increase

1168 J. VIROL.

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VOL. 10, 1972 REPAIR OF RADIATION ]

in phage sensitivity. The calculation of Freifelder(12) indicates that there is substantially more re-

pair occurring after X irradiation of T4. Hope-fully, we shall be able to isolate an X-ray-sensitivemutant of T4, unless the repair of T4 X-raydamage is intimately linked to the replication ofT4, and such a mutation would be lethal.

ACKNOWLEDGMENTS

We are grateful to Ruth Herman for technical assistance andto Michele Kissil for performing the chloramphenicol experi-ments.

This investigation was supported by a gratit from the Re-

search Foundation of the City University of New York and byPublic Health Service Research grant CA12693 from the Na-

tionai Cancer Institute.

ADDENDUM IN PROOF

A mutant deficient in the gene 32 product (DNAbinding protein) has been shown to be as ultravioletsensitive as T4x (3). We have not found this mutant(T4tsL67) to be X-ray sensitive when measured undersimilar conditions.

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