Inhibition of DNA excision by DNA polymerase-α inhibitor in UV-damaged HeLa cells

253

Toxicology Letters, 52 (1990) 253-259

Elsevier TOXLET 02343

Inhibition of DNA excision by DNA polymerase-cl inhibitor in UV-damaged HeLa cells

C.O. Joel, MA. Leel, B.S. Choi2 and K.W. Kangl ‘Department

of Biology and ‘Department

of Chemistry.

Korea Institute of Technology,

Tuejon {Republic

ofKorea] (Received 13 June 1989) (Accepted 18 February 1990) Ke.y lords: DNA polymerase; DNA excision; Aphidicolin; Dideoxythymidine

SUMMARY HeLa cells treated with 10 J/m2 of ultraviolet (UV) radiation were examined for inhibition of DNA excision by inhibitors of DNA polymerase-ct and -lp. DNA repair synthesis and excision were inhibited by aphidicolin, a specific inhibitor of DNA polymerase-ct. Decreased release of radioactive nucleotides from UV-damaged DNA by the inhibitor indicates that the action of DNA polymerase-a activity is closely associated with DNA excision. Dideoxythymidine also inhibited DNA repair synthesis but failed to block the excision, suggesting that polymerization by DNA polymerase$ might precede removal of damaged DNA.

INTRODUCTION

DNA excision repair requires a series of enzymatic reactions including the actions of endonuclease, exonuclease, polymerase and ligase. Previous studies on the enzymatic analysis of DNA repair synthesis in mammalian cells treated by ultraviolet (UV) radiation have shown that excision repair involves both DNA polymerase-cr and $3 [l-4]. It is well established that DNA repair synthesis requires base excision steps to initiate the replacement of excised nucleotides, while the relative order of the excison and repair synthesis reaction has long been a subject of controversy. The association of exonuclease activity with DNA polymerase-a in eukaryotic cells is not as evident as in prokaryotes, although exonuclease activities that degrade DNA in both the 3’ --f 5’ and 5’ --) 3’ directions have been described as one of the characterAddress ,for correspondence: CO. Joe, Ph.D., Department Taejon, 305-701, Republic of Korea.

of Biology, Korea Institute of Technology,

0378-4274/90/S 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

254

istics of prokaryotic DNA polymerase activities [5-71. However, there are several other studies indicating that eukaryotic DNA polymerases are multiprotein complexes closely associated with other nuclear enzymes including primases and exonucleases [8-l 11. In this study, we report the effects of DNA polymerase inhibitors on DNA excision in UV-damaged HeLa cells. MATERIALS

AND METHODS

Chemicals

Aphidicolin and dideoxythymidine were purchased from Sigma. MethylrHlthymidine CH-TdR, 60 Ci/m mol) and methyl[14C]thymidine (14C-TdR, 53.2 mCi/m mol) were from NEN Research Products. Cell culture

HeLa cells were grown in Eagle’s Minimal Essential Medium with 10% fetal calf serum. Cultures were initially incubated at 37°C for 2 days with 0.2 &i of 3H-TdR or 0.02 ,&i of 14C-TdR per ml before experiments were performed. Cells were rinsed with PBS and growth was arrested by placing confluent cultures in a medium containing 0.03% serum for 24 h. Cells were treated with 5 mM hydroxyurea for 1 h before exposure to UV radiation to impede replicative DNA synthesis.

A-Atlv

A- Auv

0

4 Post

Fig.

1. Release of acid-soluble

time periods

with (a-a)

3H-TMP

or without the addition

8

12

Incubation (hrsl

from UV-irradiated (A-A)

+AP

HeLa cells. After specified

5 PM aphidicolin

of trichloroacctic

(AP), the reactions

acid to 70% concentration.

post-incubation

were terminated

by

255

Measurement of repair synthesis Cultures preincubated with 0.02 PCi of 14C-TdR per ml for 2 days were irradiated with 10 J/m2 of UV radiation (254 mm) and were allowed to incorporate 3H-TdR into cellular DNA for 1.5 h in conditioned medium, with specified concentrations of inhibitors, 5 mM hydroxyurea, 10 &i/ml of 3H-TdR, and 10% fetal calf serum. Cells were rinsed with 1 x SSC twice, harvested and acid-precipitated with 2% TCA overnight and then samples were filtered onto GF filters. Each sample was washed with 5 ml of 10% cold TCA and twice with 5 ml of 95% cold ethanol. The ratio of radioactivities of 3H to 14C incorporated into insoluble material was radiometrically assayed [ 121. Measurement of excised nucleotides Cells, prelabeled in DNA with 3H-TdR (0.2 p Ci/ml), were incubated in a medium containing various concentrations of aphidicolin or dideoxythymidine for 1 h before UV radiation. Medium was removed from the culture plate and cells were rinsed twice with PBS and irradiated with 10 J/m2 of UV light (254 nm). Excised nucleotides accumulated inside cells as well as in a medium containing DNA polymerase inhibitors for the time period specifier’ ‘131. Samples of cell pellets and 10 ml of media were collected and precipitated for 16 h with the addition of solid TCA to 70% concentration, and applied to Whatman GF/C filters. The radioactivities of filtrates were counted as a measure for nucleotides excised from cellular DNA and the acid-insoluble radioactivities retained on the filter for measurement of the cellular DNA.

A-Auv A-Auv+ cwrdr 6

0 4

0

Post Fig. 2. Inhibition

of DNA

excision

were incubated

by DNA

with ( aPa)

8 Incubation

polymerase-/I

or without

(A-A)

12 (hrs)

inhibitor,

dideoxythymidine,

200 pM dideoxythymidine.

(dd Tdr). Cells

256

A

4

2

6

8

10

Aphidicolin(pM) Fig. 3. Effects of aphidicolin

on repair

IO J/m2 of UV, and co-incubated (A). Acid-soluble

3H-TMP

synthesis

released

from UV-damaged incubation

RESULTS

and exision

of DNA.

with specified concentrations

HeLa

of aphidicolin

cells were irradiated and 10 &i/ml

HeLa cell DNA accumulated

during

with

of 3H-TdR I .5 h post-

period (B).

AND DISCUSSION

This study was designed to test whether DNA excision is associated with DNA repair synthesis in which both eukaryotic DNA polymerase-cc and -j? are involved. We examined the effects of known DNA polymerase inhibitors on DNA excision in UV-damaged HeLa cells. As shown in Figure 1, acid-soluble DNA released from HeLa cells during recovery from DNA damage was significantly decreased by the addition of 5 ,uM aphidicolin. When dideoxythymidine, which is known to inhibit DNA polymerase-/I activity, was used, DNA excision was not inhibited (Fig. 2). These results suggest that HeLa cell DNA polymerase-cc might initiate the excision of damaged DNA while DNA polymerase-8 lacks an associated exonuclease activity. We next examined the effects of various concentrations of DNA polymerase inhibitors on repair synthesis and excision (Figs. 3 and 4). 10 J/m2 was used for UV radiation because this dose produced the highest level of DNA repair synthesis without

251

A

& I

I

50

too

Dideoxythymidine

I

150

t

200

(PM)

Fig. 4. Effects of dideoxythymidine on DNA repair synthesis (A) and DNA excision (B). Experimental details were same as described in Fig. 3 except that dideoxythymidine was used as inhibitor.

affecting the viability of the cells during the post-incubation period. We examined tritiated thymidine incorporation for 1.5 h into the cells as a measure of DNA repair synthesis. The results showed that there was significant inhibition of repair synthesis by aphidicolin and dideoxythymidine treatment. Replicative DNA synthesis was suppressed by the addition of hydroxyurea. The highest level of aphidicolin inhibition occurred at 3 ,BM and higher concentrations did not affect further inhibition (Fig. 3A). When dideoxythymidine, which inhibits DNA polymerase-/I more strongly than DNA polymerase-or (3, 1417), was used as inhibitor, DNA repair synthesis was similarly reduced by various concentrations (Fig. 4A). These data confirm that both DNA polymerase-cl and -B are involved in DNA repair synthesis initiated by UV damage. It is known that eukaryotic DNA polymerase-a and $2 differ in their ability to utilize template DNA with different gap sizes (18, 19). It was necessary to determine whether these two polymerase inhibitors are able to inhibit DNA excision. Acid-soluble DNA from the hydrolysis of 3H-labeled HeLa cell DNA was reduced by more

258

than 60% in the presence of 10 PM aphidicolin during repair of DNA damage (Fig. 3B). However, DNA excision was not inhibited by 200 ,uM dideoxythymidine treatment (Fig. 4B). These data suggest that the excision step in DNA repair is associated with the action of DNA polymerase-a, while polymerization by DNA polymerase-/l takes place after DNA excision. The data further imply that DNA polymerase-a polymerizes nucleotides from the site of incision coordinately with excision while polymerization precedes excision when DNA polymerase-/? is participating in gap filling. ACKNOWLEDGEMENTS

This work was supported in part by the Korea Science Foundation and by a Faculty grant from the Korea Institute of Technology, Republic of Korea. REFERENCES

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