.I. A/d. Rid. (1978) 120. 423-432

Patch Size and Base Composition of Ultraviolet LightInduced Repair Synthesis in Toluenized Escherichia coli R.BEN-I~HAI~

AND

R.SHA~~S

1)epartment of Bioloyy l’echnion-Israel Institute qf TrAnolo~qy Haifa, Ismel

Small patch repair in ultraviolet-irrudiatSed Escherich%n wli is saturat)ecI at deoxynucleoside triphosphate concentrations ( N 2 PM of each dNTP) t,hat BW severely limiting for DNA replication. The low requirement of the repair process for dNTPs permits direct demonstration of u.v.S-induced DNA synthesis by incorporation of labeled dNTP and determination of its c,xtmt, haw composition and patch size. Tt is concluded that, DNA polymeraso 1 is involved in small pat)ch repa,ir ant1 that. an awlrage of 13 t,o 16 nucleotides arc rc-insrrtcld per pyrimidinc dirrrc,r c*xcised. The average base composition of t,hc rrpairrd stwtchrs rtdjnccnt to thcx ciimers is similar to that of total E. coli DNA. .4n assay utilizing endogenous u.v.-sperific cwdonwlww to tletcrminc dirncbr casrision is described.

1. Introduction 1Jltraviolet light-induced damage in cellular DNA is correct,ed by an excision repair mechanism (Setlow & Carrier, 1964; Boyce & Howard-Flanders, 1964). Several lines of evidence indicate that in bacteria alternative pathways for excision repair exist (Hsnawalt et al., 1975; Youngs & Smith, 1973a). Before diverging, all the pathways have a common initial step which is controlled by the uvrd and uvrB genes and involves chain incision adjacent bo pyrimidine dimers in DNA (Howard-Flanders rt aZ.. 1966; Braun & Grossman, 1974; Waldstein et al.. 1974). Pathway differences seem t,o stem from the existence of different mechanisms of repair synthesis and thfb associated removal of damaged regions. Cooper & Hanawalt (1972a,B) have demonxtrat’ed that the size of repaired regions in Escherichia coli DNA varies from short’ &retches of nucleot,ides to &retches cont’aining several thousand nucleotides. Short, patch repair is believed to be mediated by DNA polymerase I and it,s associated Fi’ exonuclease activity which perform simultaneous removal of pYyrimidinc dimers and filling of gaps in parental DNA (Cooper & Hanawalt, 1972a; Kelly et al.. 1969). In mutants deficient in DNA polymerase I large patch repair predominates and is mediated by DNA polymerase II and/or III (Cooper & Hanawalt: 19726: Toungs t Smith. 19733; Masker et al.. 1973; Tait et nl.. 1974). This repair apparent]! t To whom all correspondence should be addressetl. $ Ahhrevi&ion nserl: II.~., ultraviolet, light. 123

Thus far, -DNA polgmerase 1 mediated small pat,ch repair synt#hesis has not t)(,(*u tlirectly demonstrated and t’hc extent and patch size of this repair is not known. One of the difliculties being t’he quantitation of the repair synthesis step becausch of internal pool size and re-utilization of DNA degradation products. Control of precursor and cofact’or levels can be obtained by use of permeabilized cells. St)udies with f/:. co/i made permeable to deoxgnucleoside triphosphates (dNTP) and ot’her lo\\ molecular weight, substances by treat,ment with t,oluene (Moses & ttichardson, 1970). hav(* shown that excision repair is ATP-dependent and t,hat permcabilized cells art’ ca,pabl(h of performing the incision, repair synthesis, excision and rejoining steps of t 11th repair process (Waldstein rt al., 1974: Masker & Hanawalt. 1973: Moses & Mood~~. 1975; Sharon et a.!., 1975; Reeberg & Strike, 1976; Masker, 1976; Deutsch et al., 1976). In t,his investigat,ion E. coli strains, having normal levels of DNA polpmerastb 1. were used to measure u.v.-induced DNA repair synt,hesis and to determine its pat,& size and base composition. From the dat’a presented on repair synthesis, thymints dimer excision and the disappearance of u.v.-specific endonuclease-sensit)ive sites it is concluded that in small patch repair on bhe average 13 t’o 16 nucleotides art’ IX‘inserted per pyrimidine dimer excised. Repaired stretches adjacent to t,he dimers do not contain disproport’ionat,e levels of either pyrimidim or purine nucleotid(~s.

2. Materials and Methods (a) Bacterial

strahs,

growth

co~&ions

attd prelabeli~cy

of c~elhlar

DNA

KMBL1056 (end01 thy), 1 ‘t s isogenic partner KMBLlO54 (uvrB endol thy), (uvrC thy SW bio p&A, met Zevc), KMBL1841 the UCWC proficient isogenic partner of KMBL1840, KMBL1481 (recC end01 thy met bio pheA), KMBL1479 (recB end01 thy met bio pheA) and KMBL1482 the recombination proficient isogenic partner of’ KMBL1479, were provided by Drs A. Rdrsch and I. Mattern. E. coli 1100 (endoI) and H514 (UWA endoI arg thy) were obtained from Dr Hoffmann-Berling (Diirwald & HoffmanBerling, 1968). E. coli DllO (poZA1 elzdol thy), E. co& JG139 (poZA+ thy rha ZacZ) and E. coli HMS85 (poZA+ poZB2 thy rha ZacZ Zys) were obt,ained front Dr C. C. Richardson (Campbell et al., 1972). Cultures were grown at 37°C in M9 medium (Miller, 1972) supplemented with 0.2’,‘,, (w/v) glucose, 2.5 mg Casamino acids/ml, 20 pg thymidine/ml and 0.1 pg thiamine/ml. .%mino acids and biotin when required, were added at 50 pg/ml and 1 /Lg/ml, respect.ively. To prelabel the bacterial chromosome, k3H]thymidine (20 $Zi/ml. 20 Ci/mmol) wss added to the medium and cells were grown for 18 h. E’. coli KMBL1840

(b) Toluene

treatment

and conditions

oj irradiatior~

were harvested, Stationary phase cultures grown t,o a density of -3 x lo9 cells/ml resuspended at 5 x lo@ cells/ml in 0.05 M-Tris.HCl buffer (pH 8) and treated with tolueno a8 described by Moses & Richardson (1970). For each strain, duration of treatment with toluene which resulted in maximum stimulation of ATP-dependent DNA synthesis in (,xponentially grown cells was employed. After toluenization cells were diluted with an equal volume of 0.05 M-TrissHCl buffer (pH 8), centrifuged and resuspended at 2 x log cells/ml in 0.05 M-Tris.HCl buffer (pH 8). lamp model no. R-51, For irradiation cells were exposed to 11.v. light (Mineralight; maximum emission at 254 nm) at 1.5 J/m2 per s at room temperat’urr. Since cells were’ irradiated at high concentrations, a correction for self absorption of the cell suspension was made in the calculation of incident dose.

SMALL

PATCH

425

REPAIR

(c) Standard conditions for dNTP

incorporation

Repair assay mixtures (0~15 ml) contained lOa to 2 x lOa irradiated or non-irradiated toluene-treated cells, 70 mM-Tris*HCl (pH S), 13 mM-Mgzf, 1.5 m&r-ATP, 0.5 m&r-NAD and unless otherwise indicated, 2.2 pM each of dATP, dGTP, dCTP and dTTP. The mixture was supplemented with [aH]dTTP (2.5 PCi, 45 to 50 Ci/mmol) or one of the Nuclear). After incufour [x-32P]dNTP (0.5 to 1 @i, 120 to 130 C/I mmol; New England bateion at 37’C the reaction was terminated by addition of 3 ml of ice-cold IO’j, (w/v) The acid-insoluble precipit,ate was trichloroacetic acid/O.1 M-sodium pyrophosphate. filtered through Whatman GF/C glass filters, washed 4 times with 3 ml of cold 5”,, trichloroacetic acid/ 0.1 M-sodium pyrophosphate, twice with 3 ml of 0.01 M-HCI and onct’ wit,h 3 ml of alcohol. The filters were dried and connt,ed in a toluene base scintillat,ion fluid,

(d) Isolation

of

DNA

and

thymitte

dimer nnulysl:s

DNA was isolated from irradiated and non-irradiat’ed toluene-treat,ed cells as previously described (Sharon et al., 1975), dialyzed against 0.01 M-sodium phosphate buffer (pH 6.8) and purified on hydroxyapatite columns at 60°C (Britten et al., 1974). Native DNA, acid, hydroeluting at 0.4 M-phosphate buffer, was precipitated with 10 “,A trichloroacetic lyzed with formic acid and subjected to 2-dimensional paper chromatography as described by Carrier & Set,low (1971).

(e) Sedimentation

analysis

Lysis of cells and sedimentation analysis in alkaline sucrose gradients were performed as described by Rupp & Howard-Flanders (1968). Gradients were spun at 30,000 revs/min for 90 t,o 120 min or at 9000 revs/min for 18 h at 4°C in a Beckman L-3 centrifuge with rotor no. SW50.1. For determination of the number of u.v.-specific endonuclease-sensitive sites, the number average molecular weight (M,) was calculated by computer from the distribut,ion of radioactivity in each gradient using phago T4 DNA (single-strand molecular weight of 55 x 106) as marker. (f) DNA Cellular DNA content was determined 1965) and by [3H]thymidine incorporation.

determination by the diphenylamine

reaction

(Giles

8z Myers,

3. Results (a) Repair

synthesis in toluene-treated

E. coli pal+ strains

We have previously shown that in toluene-treated and irradiated E. coli incision breaks accumulate during incubation in the presence of ATP and NAD (Waldstein et al., 1974). Such accumulation was, however, not, observed if incubation was conducted in a mixture containing, in addition to ATP and NAD, 33 pM of each dNTP (complete assay mixture). Since under the latter conditions pyrimidine dimers were excised, it was assumed that in the presence of the required cofactors and substrates excision repair occurs by consecutive action of the incision, excision, repair synthesis and rejoining steps of the repair process (Sharon et al., 1975). To determine the concentrations of dNTPs required for the repair synthesis step, toluene-t,reated and irradiated IC. coli KMBL1056 (pal+) were incubated in complete assay mixtures containing decreasing amount,s of the four dNTPs and analyzed for the formation of incision breaks in irradiated DNA. The results of Figure 1, showing that incision breaks start accumulating at concentrations below 2.2 PM of each dNTP, indicate that repair synthesis is saturated at this dNTP concentration. The low requirement of the repair process for dNTPs permitted direct demonstration of u.v.-induced DNA synthesis by incorporation of a labeled dNTP, because I6

426

H. HEN-ISHAl

AND

H. SHARON

TOP

Bottom Froctm

number

FIQ. 1. Accumulation of incision breaks in the presence of decreasing concentrations of dNTPs. Toluene-treated E. coli KMBL1056 were irradiated (30 J/m2) and incubated with ATP (1.5 mn/l), each of the 4 dNTPs: 33 PM (-O-O-), 2.2 @X NAD (0.5 mM) and the following concentrations (...~...~...),1~1~M(--~--~--),0~33~M(-~-~-),nodNTPs(-x-x-). the precursor could be used at a very high specific act’ivity and replicative synthesis is virtually eliminated at the low dNTP concentrations employed. As shown in Figure 2(a) and (b), u.v.-induced [3H]dTTP incorporation exhibits many of the characteristic properties expected of repair synthesis, it is dependent on the presence of the four dNTPs and ATP, absent in uvrA and uvrB rnutant,s (shown for uvrB) and linear with fluence up to 45 J/m2. Incorporation is proportional to the number of cells between 1 x lo* and 4 x lo8 cells per assay mixture (not shown) and optimal at 37°C (Fig. 2 (c)). Repair synthesis, as analyzed by u.v.-induced [3H]dTTP incorporation is saturated at 1.6 to 2 pM of each dNTP (Fig. 2 (cl)), which is also approximately the minimum concentration of dNTPs required to prevent, accumulation of incision breaks (compare with Fig. 1). u.v.-stimulated dTTP incorporation was observed in those E. coli strains that were neitherpoL4 nor uvr (Table 1). To determine whether DNA polymerase II participates in repair synthesis in strains having normal levels of DNA polymerase I, u.v.stimulated dTTP incorporation was determined in E. coli HMS85, a polB mutant. Table 1 shows that u.v.-stimulated dTTP incorporation occurs to a similar extent in the mutant and in its closely related wild-type strain. The results obtained with recB and recC mutants indicate that the recBC function is apparently not involved in repair synthesis in pal + strains. The absence of repair synthesis in the uvrC mutant is noteworthy, since results obtained in &vo indicate that the uvrC gene product acts subsequent to the incision st#ep by preventing resealing of incision breaks (Seeberg & Rupp, 1975). The possibility that repair synthesis in uvrC cells is not observed because incisions in this mutant are not substrates for repair enzymes has not, been eliminated. However,

SMALL

PATCH

REPAIR

Time at 37°C (mm)

o-4

437

Dose (J/m?

:((y:c 30

40

Temperature

20

VI)

I

2 [dNTP]

3

4

(4

FIG. 2. u.v.-stimulated [3H]dTTP incorporation in toluene-treated E. coli. Unless otherwise indicated toluene-treated E. co2i KMBL1056 were irradiated (30 J/n?) and incubated for 20 min at 37°C in a repair assay mixture containing ATP (1.5 mM), NAD (0.5 mnr) and 2.2 ~DI of each dATP, dGTP, dCTP and [sH]dTTP (2000 to 3000 cts/min per pmol). (a) Kinetics and dependence on ATP and dNTPs. Irradiated cells were incubated for the time indicated in the repair assay mixture (-O-O-), without ATP (--A-A--), with only [3H]dTTP (2.2 PM) (- -7 - - v - -), and 2.2 ~CMof each [3H]dTTP and dGTP (-O--C-). (b) Dose dependence of WY+ and WV- strains. E. coli KMBL1056 (uw+) (--O-O-) and E’. coli KMBL1054 (IWE) (-A-A-) were irradiated with different doses and incubated in the repair assay mixture. (c) Temperature dependence. Irradiated cells were incubated at the temperatures indicated. (d) Dependence on the concentration of dNTPs. Irradiated (-3--O --) and non-irradiated (--&--A-) cells were incubated in the repair assay mixture containing various concentrations of the 4 dNTPs. All values, except, (---A-A-) of (a), are corrected for ATP indepentlrnt C3H]dTTP incurporation.

this possibility seems unlikely since ATP and u.v.-dependent formation of incision breaks were not detected in toluene-treated uvrC cells even when polynucleotide ligase was inhibited by nicotinamide mononucleotide (not shown). Seeberg & Strike (1976) have reported similar results using plasmolyzed uwrC mut#nnts. (b) Base composition and size of repaired regions Since a repairable gap in irradiated DNA is created by excision of a pyrimidine dimer with an adjoining stretch of nucleotides, the number of repaired patches in irradiated DNA can be estimated from the number of dimers excised. To detect thymine dimer excision in toluene-treated E. coli by conventional methods (Carrier & Setlow, 1971), we have found that it is necessary to perform dimer analysis on acid hydrolysates of purified DNA rather than on hydrolysates of the total acid-insoluble cellular fraction. Another method of measuring dimer exision is to assay for the disappearance of u.v.-specific endonuclease-sensitive sites (Ganesan, 1973; Wilkins, 1973). We have modified this assay. Instead of determining sensitive sites by adding T4 or Micrococcus lutezrs u.v.-specific endonuclease t)o cell

428

R. BEN-ISHAI

AND TABLE

Repair

3a 3b

4a 4b 5a 5b

SHARON

1

synthesis in different

E. coli strains

[3H]dTTP incorporate{1 (pmol/20 min)

Strain la? lb 2a 2b 2c

R.

1.32 n.d. 1.50 1.36 1.42 1.46 1.60

KMBL1056 (w-B+) KMBL1054 (uwB) KMBL1482 (ret+) KMBL1481 (recC) KMBL1479 (recB) JG139 (poZB+) HMS85 (poZB) 1100 (pozA+) DllO (p&l) KMBL1841 (WC+) KMBL1840 (uvrC)

1.35 n.d.$ 1.45 n.d.fi

Toluene-treated E. coli non-irradiated and irradiated (30 J/m2) cells were incubated in repair assay mixture in the presence and absence of ATP. Values represent u.v. and ATP-dependent [3H]dTTP incorporation (3000 to 3500 cts/min per pmol) per 1.7 x lOa cells, corrected for nonspecific incorporation. (Non-specific incorporation was 0.1 to 0.2 pmol in end01 mutants and 0.3 to 0.4 pmol in endoI proficient cells.) n.d., non-detectable ( excised per 1.5 ,ug DNA. To calculate the patch size from the disappearance of u.v.-specific endonuclease-sensitive sites (b), sites initially present were taken from dimer analysis (+12’?; A PyT/T for 30 J/m2 and 0.06% P$&/T for 15 J/m2).

The base composition and the total amount of dNTPs incorporated during repair was obtained by assaying for u.v.-induced incorporation of each one of the four [a-32P]dNTPs. Table 3 shows that dTTP and t,o a small extent dCTP are preferentially incorporated during repair synthesis. However, by taking into account that for each dimer excised two pyrimidines are preferentiallyinserted, it. is found t.hat theaverage base composition of the stretches adjacent to t’he dimer is similar t’o that of total E. coli DNA (Lehman et al., 1958).

4. Discussion Our experiments demonstrate that, in toluene-permeabilized E. coli (poE+ ) repair synthesis is dependent on ATP and functional uvrd, uvrB, uvrC genes, and is saturated at concentrations of dNTPs (~2 PM of each dNTP) which are limiting for DNA replication. The absence of repair synthesis in uvrA and uvrB mut’ants is apparently due t’o their inability to perform the incision step of repair. It is as yet, not clear if the uvrG

I