Proc. Nati. Acad. Sci. USA

Vol. 75, No. 6, pp. 2598-2602, June 1978 Biochemistry

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts (excision repair/pyrimidine dimer)

CHARLES ALLEN SMITH AND PHILIP C. HANAWALT* Department of Biological Sciences, Stanford University, Stanford, California 94305

Communicated by R. B. Setlow, March 3, 1978

ABSTRACT The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [3Hlthymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg2+dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in W138 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.

MATERIALS AND METHODS Cell Culture and 32P Prelabeling. Those techniques were as described (9) except for XP12BE cells, from E. Friedberg, grown in minimal essential medium with 20% fetal calf serum (Gibco). Stocks of W138 and XP12BE were discarded when cells no longer grew rapidly to confluence; three frozen ampoules of WI38 and two of XP12BE were reconstituted during the study. Preparation of Nuclei. BrdUrd (to 10 MM) and FdUrd (to 1 uM) were added 2 hr before cells were washed and irradiated (9). Where desired, cells were reincubated in reserved medium The 100-mm plates were then rinsed three times with 5 ml and incubated for 10 min at 40 in 3 ml of buffer A (5 mM MgCl2/1 mM EDTA/6 mM 2-mercaptoethanol/10 mM Tris-HCI, pH 8 at 200). Two milliliters was removed, and the monolayer was scraped down, pipetted against the plate four to five times with the reserved buffer, and held at 00. Preparations from imirly treated plates were mixed and centrifuged in 12-ml conical glass tubes at 2500 rpm in an International UV centrifuge, 200, for 3 min. The pellets were resuspended in fresh buffer A by brief Vortex mixing. Assay. To the resuspended nuclei (generally ca 5 X 106 per assay, from two plates) were added enzymes or their buffers and a substrate mix, prepared just prior to the experiment. [3H]dTTP (New England Nuclear, ca 50 Ci/mmol) in 50% EtOH was evaporated under a stream of N2 and resuspended in 20 mM MgCl2/0.3 M NaCl/0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), and other constituents, stored frozen in this buffer. Final concentrations in the 0.5-iml assay were 7 mM Tris, 10 mM MgCl2, 0.1 M NaCl, 4 mM 2-mercaptoethanol, 0.7 mM EDTA, 5 mM ATP, 0.1 mM each dATP, dCTP, and dGTP (P-L Biochemicals), 10,uM BrdUTP (Sigma), 30 mM Hepes, and 2MgM [3H]dTTP (100 uCi/ml), pH 7.8 at 370. When 100 ,l of endonuclease V from coliphage T4-infected cells (T4 endo V) was added, the MgCl2, NaCl, and Hepes concentrations were reduced by 20%. Tubes were incubated at 370, finger vortex mixed about every 10 min, and chilled. In kinetics experiments, EDTA was added to 50 mM. Nuclei were pelleted as above for 1 min, resuspended in 2.5 ml of 10 mM Tris/10 mM EDTA/0.5% sodium dodecyl sulfate/50 ,Mg of Proteinase K (EM Labs, Elmsford, NY) per ml, and incubated for 2 hr at 500 or overnight at 37°. The digests were passed twice through a 25 gauge needle, and the parental Abbreviations: XP, xeroderma pignentosum; T4 endo V, endonuclese V from coliphage T4-infected cells; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

Excision repair of damaged DNA is thought to proceed by removing a stretch of nucleotides containing the damage and then replacing that segment by repair replication, utilizing the intact

complementary strand as template (1). Skin fibroblasts from patients with "classical" xeroderma pigmentosum (XP), a disease characterized by high susceptibility to sunlight-induced skin malignancies, are extremely UV sensitive, and they exhibit reduced levels of pyrimidine dimer removal and repair replication (2, 3). It was originally suggested (4, 5) that the defect in XP was the absence of a putative endonuclease to initiate the excision repair process. However, XP cell lines distribute into at least five complementation groups (3), and extracts from XP cells can promote release of dimers from DNA (6). The study of repair replication in cells made permeable by toluene has provided insight into the details of that process in bacterial systems (7, 8). We sought to develop a system in human cells to which various "repair enzymes" and the immediate precursors for DNA synthesis might be introduced. Initial characterization of such a system is reported here. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

*

2598

To whom reprint requests should be addressed.

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Smith and Hanawalt density DNA was isolated in neutral CsCl gradients and analyzed in alkaline CsCl gradients (9). The position of parental density DNA in the neutral gradients was determined from the 32P radioactivity in aliquots of the fractions spotted onto paper strips and assayed in a Packard scintillation spectrometer with Omnifluor (New England Nuclear) in toluene. To assay 3H radioactivity, the DNA in these aliquots was precipitated with C13CCOOH and collected on Millipore filters (HAWP) that had been soaked in 0.2 M sodium pyrophosphate. Alkaline CsCl gradients were fractionated into tubes containing 10 ,g of carrier DNA. The 3H/UP ratio over the four to five peak fractions of normal density DNA is the value used for repair replication. Enzymes. T4 endo V was prepared by P. Seawell by a modification of the procedure of Friedberg and King (10). Twenty microliters of preparation I or 100 JAl of the less concentrated preparation II, or its buffer [50 mM Tris/10% (wt/vol) ethylene glycol, pH 8]- was used per 0.5-ml assay. DNase I (Sigma, DN-C) was made to 1 mg/ml in 10 mM Tris and frozen in several aliquots, which were thawed and diluted with buffer A just before use. Alkaline Sucrose Gradient Analysis. Cells were grown to confluence in 4MiM dThd, 2 MACi of [3H]dThd per ml. About 104 cells or nuclei in 50 M1 of 10 mM Tris/10 mM EDTA were layered onto a 0.2-ml layer of 1 M NaOH/50 mM EDTA, placed atop linear 5-20% (wt/vol) sucrose gradients containing 2 M NaCl, 0.3 M NaOH, and 5 mM EDTA. After 3 hr in the dark, the gradients were centrifuged for 2 hr at 30,000 rpm, 200, in a Spinco SW 50.1 rotor, and fractionated onto strips of Whatman no. 17 paper, which were washed with Cl3CCOOH and assayed for radioactivity. Number-average molecular weights were calculated (l1), with reference to "4C-labeled T2 phage DNA (65 X 106). Purified DNA was centrifuged at 45,000 rpm for 4 hr, with unit length ColEl DNA (2.1 X 106) as reference. Sonicated DNA was centrifuged at this speed for 15 hr. and the gradient was calibrated with a HindIII digest of closed circular simian virus 40 DNA (12). Length Analysis of Synthesized DNA. A plastic 12 X 75 mm tube, in ice water, containing 0.6 ml of DNA in 10 mM Tris/1 mM EDTA was held in a clamp so that the bottom of the special micro tip of the Branson 140 sonifier was at the center of the liquid. The sonifier was run at a setting of 4, for 6-10 intervals of 30 sec, spaced by 30-sec intervals in which the tube contents were mixed. Alkaline CsCl gradients (3 ml) containing 0.1 M K2HPO4 titrated to pH 12.5 were adjusted to a refractive index of 1.4060 and centrifuged in polyallomer tubes in the SW 50.1 rotor at about 220, 35,000 rpm for 90 hr (sonicated DNA) or 40 hr (unsonicated DNA). RESULTS We have adapted the radioisotope and density labeling method for determination of repair replication (7, 9, 13) to our in vitro assay, which is that described by Seki et al. (14) modified for the inclusion of BrdUTP. It requires that lengths of hybrid DNA synthesized by semiconservative synthesis be long enough to be effectively separated from DNA of parental density, because repair is defined as synthesized stretches short enough to alter only slightly the density of the DNA containing them. In rapidly growing VA13 cells this condition was met with our assay conditions. However, to minimize the possibility that some aberrant semiconservative synthesis in vitro might appear as repair, we used contact-inhibited cells, in which very little semiconservative synthesis occurs. Our in vitro system is prepared rapidly without trypsinization and closely resembles a Dounce homogenate of cells

2599

-

0

-6

1 0

oq

0

x

V-

B

Endo V -6+UV

CL

XE isa

D+UV +Endo V

10

4

2 -5 5 10 15 20 25 5 10 15 20 25 Fraction FIG. 1. Repair replication in W138 nuclei. Radioactivity profiles of alkaline CsCl gradients of purified normal density DNA. UV irradiation (30 J/m2) was 30 min prior to preparation of nuclei. Incubation was 20 min. T4 endo V was preparation I. Centrifugal field increases toward the left. Points omitted were at background. A, 32p prelabel; 0, 3H from [3H]dTTP in assay.

swollen in Mg2+-containing buffer. In the XP12BE preparations the nuclei are not as free of cell constituents and are more likely to clump and adhere to glass than are the nuclei in preparations from the other cell lines. However, all the DNA in these preparations is susceptible to attack by DNase I and T4 endo V. Repair Replication in Isolated Nuclei. In nuclei from W138 cells, essentially no incorporation of [3H]dTTP occurs in normal density DNA, but such incorporation is found in nuclei from cells UV irradiated (30 J/m2) 30 min before the preparation of nuclei (Fig. 1 A and B). In marked contrast, nuclei from similarly treated XP12BE cells show no UV-stimulated incorporation (Fig. 2 A and B). These cells performed essentially no repair replication in 2.5 hr in our whole cell assay (10). [Repair replication in nuclei from rapidly growing simian virus 40 transformants of WI38 and XP12RO (group A) followed the same pattern, with higher backgrounds in the nuclei from unirradiated cells.] With a single set of WI38 cells, repair replication in nuclei and in whole cells was compared. In whole cells, repair incorporation of Br[3H]dUrd was linear with time for the first 2 hr

0

I

t

0 T-

0

x

x

E

E

0

0

m

5 10 15 20 10 15 20 Fraction FIG. 2. Repair replication in XP12BE nuclei. Details are identical to those of Fig. 1. Larger fractions were collected.

5

2600

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Smith and Hanawalt

after irradiation, with about a 5-min lag. The rate of incorporation of 3H per 32P prelabel for the whole cells was corrected by the ratio of the final specific activity of Br[3H]dUrd used in that assay to that of the [3H]dTTP/BrdUTP mix used in the assay of nuclei. Assuming that the incorporation in nuclei occurred over a 5-min period (see below), it represented 1.2 times the corrected rate in whole cells. In a single experiment in which lysates were centrifuged directly in alkaline CsCl gradients (13), the incorporation in WI38 nuclei after 5 J/m2 was approximately 65% of that after 10 or 30 J/m2. In the above experiments the nuclei were from cells already engaged in repair replication. In two separate experiments in which cells were irradiated just prior to scraping the monolayer, repair incorporation was 33% and 35% of that in nuclei from cells irradiated 1/2 hr prior to nuclei preparation. A value of 38% was obtained when a preparation of nuclei was spread on a petri plate and irradiated just prior to centrifugation.

Stimulation of Repair Replication by UV-Specific Endonuclease. When T4 endo V was included in repair replication assays, a marked increase in incorporation was seen in nuclei from irradiated W138 cells (Fig. 1D) and incorporation was observed in nuclei from irradiated XP12BE cells (Fig. 2D). The enzyme did not stimulate incorporation in nuclei from unirradiated cells (Figs. 1C and 2C), nor did it stimulate incorporation into -DNA of hybrid or intermediate density as judged from the neutral CsCl gradients. When T4 endo V was included in a 20-min incubation, incorporation in nuclei from W138 cells irradiated just before disruption was 87% of that in nuclei from preirradiated cells. A value of 86% was obtained in a similar experiment with XP12BE cells. Accessibility of DNA to Endonucleases. Nuclei from 3H-prelabeled cells were mock assayed and their DNA was analyzed on alkaline sucrose gradients. Single fast sedimenting components with number-average molecular weights (Mn) of 200-250 X 106 were observed with nuclei from unirradiated cells of both types, after both 3- and 15-min incubations. Profiles from nuclei of irradiated cells were broader, with Mn values around 90-130 X 106. Single slowly sedimenting components were observed from nuclei of irradiated cells incubated with T4 endo V, preparation I for 3 min (Mn = 4-5 X 106) or preparation H for 15 min (7-10 X 106). These molecular weights are very approximate because the components moved only a short distance, but are consistent with nicking at 10-20% of the pyrimidine dimers induced by the UV. The T4 endo V did not reduce the molecular weight in nuclei of unirradiated cells. DNase I (solution B) as used in subsequent repair assays reduced Mn values to 20 X 106 (WI38) and 6 X 106 (XP). Single components were again observed. Effect of Added DNase I. It was plausible that the effect of T4 endo V was due to nonspecific synthesis occurring at nicks introduced by this enzyme that occur only on irradiated DNA (15). Further, the T4 endo V preparations might have been contaminated with other enzymatic activities responsible for the synthesis. To test the specificity of the T4 endo V results, assays were done including DNase I (Table 1). Both enzymes produce nicks with 5'-hydroxyl and 3'-phosphate groups (16, 17). In WI38 nuclei DNase I promoted a small increase in incorporation which was minor compared to increases due to UV irradiation or T4 endo V. Because DNase itself did not decrease synthesis in those cases in which synthesis was observed in its absence, the DNase activity was not so great as to degrade DNase-promoted synthesis. In XP12BE nuclei, DNase I was included at a higher concentration, but the incorporation pro-

Table 1. Effect of DNase I and T4 endo V on repair replication in nuclei Repair replication Exp. III Exp. II Exp. I Experimental conditions UV T4 endo V DNase I (WI38) (WI38) (XP12BE) -

-

-

+ + + +

+

+ + + +

+

+ +

2 -

5 5

4 5

9

18

51

15 62

40

92 186 112 200

160 84 200

5 230 77 180

Values for repair are the 3H to 32P ratios X 100 for each experiment and are not normalized for comparison between experiments. Experiment I included T4 endo V preparation I and DNase solution A (2 ,g/ml). Experiments II and III included T4 endo V preparation II and DNase solution B (5 ug/ml for WI38, 10 ,ug/ml for XP).

moted compared to that by T4 endo V was still modest. T4 endo V did not stimulate synthesis in nuclei from unirradiated cells also treated with DNase I. Size of Lengths Synthesized. Nuclei from irradiated W138 and XP cells were incubated with T4 endo V for 60 min. Sonication of portions of the purified parental density DNA from these nuclei reduced the average fragment length to about 225 nucleotides, estimated from the positions of the centers of bands of DNA centrifuged in alkaline sucrose gradients, with reference to the position of the 216-nucleotide-long simian virus 40 HindIII K fragment (W. Fiers, personal communication). For control samples, the centers of mass of the 3H distributions in alkaline CsCl gradients were shifted to the dense side of the centers of mass of the 32P distributions by 0.1 and 0.17 fractions for the WI38 and XP DNA (Fig. 3). For sonicated samples the II

A 4 3

2

Control 3

_ _

-

1l

2:ER

I

_

2

_ .~~~~~ ,~~~~~~~ 1

II1

1

y

-Z R~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ x

x

E 0.

CL

0.

CL m

15 Fraction

FIG. 3. Analysis of T4 endo V-stimulated synthesis in XP12BE nuclei. Radioactivity profiles of parental density DNA centrifuged in alkaline CsCl. (A) DNA as recovered from neutral CsCl gradient; (B) DNA after sonication. Fraction 15 was incompletely recovered. Centrifugal field increases toward the left. A-A, 32p prelabel; 0 --- 0,

3H

repair label.

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Smith and Hanawalt 2

-4~~~~~~~~~4

cu

r._ A3

0

0

._

co

d

._

w

6 A

3 2

11 0

10

20 30 Time, min

40

FIG. 4. Kinetics of repair replication in WI38 and XP12BE nuclei. (A) All experiments included a 15-min point, to which data are normalized. 0, WI38 nuclei, T4 endo preparation IL Point near zero is unirradiated control. A, XP nuclei, same details. 0, W138 nuclei, incubated with T4 endo V preparation II in absence of Mg2+ at 200 10 min prior to addition of assay components and incubation. Aliquots of a single assay mix were withdrawn at times indicated. v, XP nuclei, as for not enough remained for 60-min determination. (B) Repair replication was normalized to value for 10 min in absence of T4 endo V, except for that with XP nuclei, in which no incorporation is observed in absence of the enzyme; for XP nuclei the values were normalized to that for 15 min, which was plotted on the line formed by the experiment with WI38 nuclei. WI38 nuclei, no T4 endo V. Point near zero is for unirradiated control. *, W138 nuclei, no T4 endo V; normalization point omitted. 0, WI38 nuclei, T4 endo V preparation I included, same experiment as M. 0, WI38 nuclei, at 10 min, 400 Ml of T4 endo V preparation II was added to the single assay mixture. XP nuclei, T4 endo V preparation II included except in point near zero, where enzyme buffer was added.

57

60

preparation.

0,

A,

shifts were 1.5 and 1.6 fractions. Few or no very long stretches observed. A shift of 12.3 fractions was found for unsonicated hybrid DNA synthesized in nuclei of unirradiated cells (Table 1, experiment III). These data, corrected for the dilution of the BrdUTP by dTTP, lead to an estimate of about 35 nucleotides for the average length of the stretches. An experiment in which [3H]dATP replaced the [3H]dTTP confirmed that the stretches are short. were

Table 2. Effects of modifications of the assay system on repair replication in WI38 nuclei

Complete System -ATP 2 X [ATP]

_-Mg2+

2 X [Mg2+] - dNTPs

Relative activity* + T4 No enzyme endo V

100 54

100 10 75

0

26 140 2-Mercaptoethanol + 10% ethanol 14 1 [3H]dTTP + [3H]dThd + 1 mM N-ethylmaleimidet 33 + 0.1 mM p-chloromercuribenzenesulfonic acid 22 * Values are 3H/32P relative to those for the standard assay, for each column, which has been set to 100. t In this experiment, nuclei were incubated with T4 endo V in the absence of Mg2+ prior to assay to ensure that the compound did not inhibit the endonuclease. -

-

Kinetics of Repair Replication in Nuclei. (Fig. 4.) The addition of T4 endo V to WI38 nuclei extends the period of incorporation from 5 min to over 1 hr. When the enzyme was added to nuclei at 10 min, synthesis resumed. As above, we compared the incorporation rate in WI38 whole cells to the rate in nuclei to which T4 endo V was added, measuring the difference in repair for 15- and 30-min incubations. In whole cells it was found to be about 2.4 times that in nuclei, in reasonable agreement with the earlier comparison, and with the kinetics of incorporation. Incubation of nuclei with T4 endo V (10 min at 20°) before addition of Mg2+ and substrate allows nicking to precede synthesis with minimal loss of total activity (13% in a 10-mi assay). Kinetics of repair synthesis using this protocol were similar to those obtained previously (Fig. 4B). Substrate Requirements. (Table 2.) Synthesis in W138 nuclei is stimulated by added ATP and dNTPs. The T4 endo V enhancement (a factor of 3 for the 30-min incubation used here) is dependent upon ATP and the dNTPs, because the synthesis observed in their absence is roughly that synthesized in 5 min without added enzyme.

DISCUSSION The repair replication we observe in nuclei from contact-inhibited normal cells is strictly UV dependent and is not observed in nuclei from group A XP cells, strong evidence that the synthesis reflects repair rather than aberrant replication enhanced by UV. The synthesis is about that expected for a similar period of repair synthesis in whole cells. This is a rough comparison, because specific activities of precursors in cells are difficult to determine, some dilution of [3H]dTTP in nuclei by residual levels of BrdUTP is expected, and we do not know the kinetics within the initial 5-min period. It is likely that some of the incorporation is due to the completion of repair replication initiated in whole cells. However, repair replication can be initiated in the nuclei themselves at reduced levels. The rapid termination of synthesis is not due to exhaustion of precursors or cofactors or degradation of synthetic capacity, because addition of T4 endo V after 10 min caused it to resume. We suggest that it is due to exhaustion of specific nicks at or near UV photoproducts. The human UV endonuclease(s) may rapidly diffuse out of the nuclei or may not be presented with optimal conditions in our assay. Preirradiated cells probably contain more of these specific nicks than cells irradiated just before disruption, if such nicks made in viwo are stabilized at the low temperature at which the nuclei are prepared. Cells irradiated after freezing and thawing, another method of rendering them permeable (A. van Zeeland, C. A. Smith, and P. C. Hanawalt, unpublished data), showed no repair synthesis, suggesting that freezing may inactivate the cellular UV endonuclease(s). However, cells irradiated 30 min before freezing and thawing showed significant synthesis, although less than that in nuclei, suggesting that in nuclei from preirradiated cells specific nicks are made both before and for a limited time after

o,

Assay conditions

2601

Incubation of nuclei from irradiated normal cells with T4 endo V resulted in a large extension of the period of synthetic activity, which was dependent on ATP and deoxynucleoside triphosphates. The cessation of activity in this case may be due to exhaustion of precursors or gradual diffusion out or degradation of essential cellular components. It is not due to exhaustion of nicks near pyrimidine dimers, because the amount of incorporation observed is consistent with 35-nucleotide stretches synthesized at only about 5% of the nicks introduced.

2602

Proc. Nati. Acad. Sci. USA 75 (1978)

Biochemistry: Smith and Hanawalt

Synthetic activity with similar inetics was observed in nuclei from irradiated XP12BE cells exposed to T4 endo V, suggesting that these cells contain the same polymerizing activities as the normal cells but lack the ability to produce proper substrate nicks near UV photoproducts. Comparison of the incorporation per cell between XP12BE and W138 is difficult, because the XP preparations contain more cytoplasm around the nuclei and are not as dispersed as W138 preparations. However, the overall levels of incorporation observed for roughly the same number of cells are comparable. Tanaka et al. (18) demonstrated T4 endo V stimulation of [3H]dThd incorporation (unscheduled-DNA synthesis) in UVirradiated XP cells made permeable by Sendai virus. Our results confirm this observation, and we have been able to investigate biochemical aspects of this synthesis in more detail. The T4 endo V-mediated synthesis in both XP and WI38 cells is in the short stretches characteristic of repair replication in whole cells (19; our unpublished results). The perceptible shift in density of the short fragments containing repair label is evidence that the incorporation is not due to addition of only one or a few nucleotides at the nicks produced by the endonuclease. This is also indicated by the requirement for the unlabeled deoxyribonucleoside triphosphates in the assay. We have not determined whether the synthesized stretches are ligated at both ends to parental DNA. The absence of significant DNase I enhancement of synthesis -indicates that the stimulation by T4 endo V must be more specific than introduction of random nicks into the DNA. (The DNA molecular weight after 15 min of incubation with DNase I shows that the nicks are not rapidly sealed in the nuclei.) It is likely that a nick at or near damage in the DNA is a favored substrate for repair synthesis, possibly due to extensive local melting in this region, rendering the DNA configuration favorable for exonuclease or polymerase activities. The small amount of synthesis stimulated by DNase I may be occurring at those nicks appearing in a favored configuration for other reasons. If the synthesis results from some enzymatic activity in the endonuclease preparation, then that must require the same favored configuration. Our T4 endo V preparations do not contain sufficient exonuclease activity to release pyrimidine dimers from purified DNA (U. K. Ehmann, personal communication). The sensitivity of the repair synthesis in nuclei to sulfhydryl group blocking agents suggests that a large portion of it may be carried out by either the a or 'y polymerase, because the (3 polymerase is relatively insensitive to these reagents in vitro (20). It has been shown that T4 endo V can replace a missing dimer-specific endonuclease activity in uvr mutants of Escherichia coli (21). Our studies lend support to the notion that the defect in XP (group A) also involves the production of endonucleolytic cleavages near damaged sites in DNA. Just as with E. coli, this defect is not limited to the inability to recognize pyrimidine dimers, because XP cells are sensitive to a variety of agents that damage DNA-e.g., 4-nitroquinoline 1-oxide (22). Tanaka et al. (23) have reported that unscheduled synthesis is not restored in XP cells treated with 4-nitroquinoline 1-oxide and made permeable to T4 endo V, presumably because the enzyme does not recognize the lesion produced. Extracts of XP12BE cells have been reported to contain an activity that promotes dimer excision from purified DNA but not from unfractionated chromatin (6). Thus the defect in these

cells could involve access of endogenous endonuclease activity to pyrimidine dimers in chromatin, a problem not encountered by the T4 ,endo V. The use of isolated nuclei or other permeable cell systems may allow further elucidation of the details of excision repair in human cells and an analysis of the defects in repair in human genetic disease. We thank N. Baker for technical assistance, R. Arrabal for general laboratory support, and A. Sarasin for simian virus 40 DNA. We are especially indebted to P. Seawell for her generosity in supplying the endonuclease V. These studies were supported by grants NP 161 from the American Cancer Society and GM 09901 from the National Institute of General Medical Sciences. C.A.S. wishes to dedicate this work to the memory of his father, Charles Henry Smith, who lost his personal battle with cancer while these studies were in progress. 1. Hanawalt, P. C. (1975) in Molecular Mechanisms for Repair of DNA, eds. Hanawalt, P. C. & Setlow, R. B. (Plenum Press, New York), pp. 421-430. 2. Cleaver, J. E. (1968) Nature 218,652-656. 3. Robbins, J. H., Kramer, K. H., Lutzner, M. A., Festaff, B. W. & Coon, H. G. (1974) Ann. Intern. Med. 80,221-248. 4. Cleaver, J. E. (1969) Proc. NatI. Acad. Sci. USA 63,428-435. 5. Setlow, R. B., Regan, J. D., German, J. & Carrier, W. L. (1969) Proc. Nati. Acad. Sci. USA 64,1035-1041. 6. Mortelmans, K., Friedberg, E. C., Slor, H., Thomas, G. & Cleaver, J. E. (1976) Prac. Nati. Acad. Sci. USA. 73,2757-2761. 7. Masker, W. E. & Hanawalt, P. C. (1973) Proc. Natl. Acad. Sci. USA 70, 129-133. 8. Hanawalt, P. C., Cooper, P. K., Burrell, A.-& Masker, W. E. (1975) in DNA Synthesis and Its Regulation, eds. Goulian, M. & Hanawalt, P. (W. A. Benjamin, Menlo Park, CA), pp. 774-790. 9. Smith, C. A. & Hanawalt, P. C. (1976) Biochim. Biophys. Acta 447, 121-132. 10. Friedberg, E. C. & King, J. J. (1971) J. Bacteriol. 106, 500507. 11. Rupp, W. D. & Howard-Flanders, P. (1968) J. Mol. Biol. 31,

291-305. 12. Fiers, W., Rogiers, R., Soeda, E., Van de Voorde, A., Van Heuverswyn, H., Van Herreweghe, J., Volckaert, G. & Yang, R. (1975) FEBS Symp. 39, 17-33. 13. Smith, C. A. & Hanawalt, P. C. (1976) Biochim. Biophys. Acta 432, 33-347. 14. Seki, S., Le Mahieu, M. & Mueller, G. C. (1975) Biochim. Biophys. Acta 378,333-343. 15. Simon, T. J., Smith, C. A. & Friedberg, E. C. (1975) J. Biol. Chem.

250,8748-8752. 16. Minton, K., Durphy, M., Taylor, R. & Friedberg, E. C. (1975) J. Biol. Chem. 250, 2823-2829. 17. Hudson, B., Upholt, W. B., Devinny, J. & Vinograd, J. (1969) Proc. Natl. Acad. Sci. USA 62,813-820. 18. Tanaka, K., Sekiguchi, M. & Okada, Y. (1975) Proc. Natl. Acad. Sci. USA 72,4071-4075. 19. Edenberg, H. & Hanawalt, P. (1972) Biochim. Biophys. Acta 272,

361-372.

20. Weissbach, A. (1977) Ann. Rev. Biochem. 46,25-47. 21. Taketo, A., Yasuda, S. & Sekiguchi, J. (1972) J. Mol. Biol. 70, 1-14. 22. Ikenaya, M., Ishii, Y., Tada, M., Kokonaga, T., Takebe, H. & Kondo, S. (1975) in Molecular Mechanisms for the Repair of DNA, eds. Hanawalt, P. C. & Setlow, R. B. (Plenum Press, New

York), pp. 763-771.

23. Tanaka, K., Hayakawa, H., Sekiguchi, M. & Proc. Natl. Acad. Sci. USA 74,2958-2962.

Okada, Y. (1977)

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts.

Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2598-2602, June 1978 Biochemistry Phage T4 endonuclease V stimulates DNA repair replication in isolat...
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