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The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens
231
Mutation Research, 183 (1987) 231-239 DNA Repair Reports Elsevier MTR 06218
The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens Kazuyuki Tao, Asao Noda * and Shuji Yonei Laboratory of Radiation Biology. Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606 (Japan) (Received 25 October 1986) (Revision received 16 December 1986) (Accepted 17 December 1986)
Keywords: Micrococcus luteus mutants; Excision-repair mechanisms; 4-Nitroquinoline 1-oxide; Mitomycin C; Cis-platinum; 8Methoxypsoralen, Angelicin; Endonuclease activity.
Summary M. luteus mutants showing increased sensitivity to both UV and 4-NQO were isolated after the treatment of parental ATCC4698 strain with MNNG. The mutants were also highly sensitive to mitomycin C, cis-platinum, 8-methoxypsoralen (8-MOP) plus near-UV and angelicin plus near-UV in various degrees. The endonuclease activity specific for pyrimidine dimers in UV-irradiated DNA was normally detected in extract of the mutants. With regard to host-cell reactivation ability the mutants fell into two groups. The hcr- mutants lacked the ability to reactivate UV-damaged N6 phage and were resistant to X-rays. The incision of DNA did not occur during incubation after the treatment with angelicin plus near-UV in the hcr- mutants, whereas it occurred in the parental strain. The facts indicate that the hcr- mutants are defective in the incision mechanism which has a wide substrate specificity, similar to the UVRABC nuclease of E. coli. On the other hand, the incision of DNA and the removal of UV-induced thymine dimers from DNA occurred in the hcr- mutants as well as in the parental strain, which is ascribed to the UV endonuclease activity. Compared with the hcr- mutants, hcr ÷ mutants were highly sensitive to X-rays, like r e c A - mutants of E. coli.
The major photoproducts produced in DNA upon exposure to UV are the cyclobutane dimers and the (6-4) photoproducts formed at adjacent pyrimidine bases (Haseltine, 1983). In E. coli, the photoproducts are recognized by a complex of the products of three uvr + genes (uvrA +, uvrB + and uorC +) (Sancar and Rupp, 1983; Yeung et al., * Present address: Meiji Institute of Health Science, 540, Narita, Odawara 250 (Japan). Correspondence: Dr. Shuji Yonei, Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606 (Japan).
1983; Franklin and Haseltine, 1984). The UVRABC nuclease can also recognize DNA damage induced by 4-NQO, bifunctional psoralen plus near-UV, mitomycin C, cis-platinum and monofunctional angelicin plus near-UV (Ikenaga et al., 1975; Cole et al., 1976; Alazard et al., 1982; Sancar and Rupp, 1983; Sancar et al., 1985). Mutants of E. coli defective in one of the uor + genes show increased sensitivity to these agents (Kondo et al., 1970; Hanawalt et al., 1979). The enzymatic activity specific for UV-irradiated DNA has been purified from M. luteus (Carrier and Setlow, 1970; Nakayama et al., 1971;
0167-8817/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
232 Riazuddin and Grossman, 1977). A detailed characterization of the mechanism of action of the M. luteus endonuclease has shown that it cleaves the N-glycosylic bond between the 5'-pyrimidine of a dimer and deoxyribose moiety and then cleaves the phosphodiester bond on the 3'-side of the resulting baseless deoxyribose residues (Haseltine et al., 1980; Gordon and Haseltine, 1980). The M. luteus UV endonuclease is specific for pyrimidine dimers in UV-irradiated DNA (Haseltine et al., 1980). The UV endonuclease has been shown to be involed in in vivo excision repair of pyrimidine dimers in M. luteus (Belle and Linn, 1982). However, a mutant of M. luteus defective in the UV endonuclease activity showed almost the same sensitivity to UV as the wild-type strain (Mahler et al., 1971; Takagi et al., 1968; Riazuddin et al., 1977). On the other hand, a mutant of M. luteus which shows increased sensitivity to UV has normal level of the UV endonuclease activity (Mahler et al., 1971; Zherebtsov and Tomilin, 1982). Thus, the level of endonuclease activity specific for pyrimidine dimers seems not to correlate with the sensitivity of M. luteus to UV. Furthermore, the UV endonuclease cannot recognize D N A damages induced by cis-platinum and psoralen plus nearUV (Fraval et al., 1978; Sancar and Rupp. 1983). From the facts it is possible that M. luteus has another type of incision mechanism which has a wide substrate specificity like the E. coli UVRABC nuclease in addition to the UV endonuclease. If this is the case, a mutant which cannot repair bulky D N A damage induced by UV and various chemical mutagens would be isolated. To test the possibility, we attempted to isolate mutants which show increased sensitivity to both UV and 4-NQO. This paper shows that M. luteus has two different incision mechanisms responsible for DNA damage. Materials and methods
Bacteria and phage The parental ATCC4698 strain and a UV-sensitive strain G7 (Riazuddin et al., 1977) of M. luteus and N6 phage used in this study were kindly supplied by Dr. H. Nakayama, Kyushu University. The ceils were grown at 3 4 ° C in LB broth (10 g bactotryptone [Difco], 5 g yeast extract
[Difco], 5 g NaCI and 1 g glucose in 1000 ml of distilled water, pH 7.2).
Chemicals cis-Diamminedichloroplatinum(II) ( cis-platinum) was a generous gift from Dr. M. Ikenaga, Kyoto University. 4-Nitroquinoline 1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) and mitomycin C were purchased from Wako Pure Chemicals (Osaka). 8-Methoxypsoralen (8-MOP) and angelicin were obtained from Polyscience Inc. (U.S.A.). Colicine E1 (ColE1) D N A was the product of Takara Shuzo (Kyoto). [6-3H]Thymidine was obtained from New England Nuclear (U.S.A.). Isolation of mutants Mutants were isolated after the treatment of parental ATCC4698 strain in stationary phase with 30 / , g / m l of M N N G at 30°C for 30 min. The treated cells were washed twice, resuspended in the original volume of LB broth and then shaken at 34°C for about 15 h. The cells were plated on LB agar and incubated at 34°C for 3 days. UV (250 J/mZ)-sensitive mutants and 4-NQO (100 /,g/ml)-sensitive mutants were isolated separately by replica plating method. Survival experiments with radiation The cells in stationary phase were washed, resuspended in phosphate-buffered saline (PBS, pH 6.5) and then irradiated with UV from germicidal lamps (15-W × 2) at a dose rate of 2 J/mZ/sec, estimated using a UV intensity meter (UVR254, Topcon). X-Irradiation was performed with a Toshiba X-ray machine (KXL-19-2) operating at 180 KVp and 20 mA. The dose rate was 400 r a d / m i n which was measured by the Fricke dosimeter. Following irradiation, the cell suspensions were diluted appropriately and plated on LB agar (2% agar). After incubation at 34°C for about 3 days viable colonies were counted to estimate the survival. Survival experiments with chemicals The cells in stationary phase were washed, resuspended in PBS. The suspensions were kept in the dark at 3 0 ° C for 1 h after the addition of
233 mutagens at various concentrations. The survival estimation was performed as described above.
thymine-containing dimers was performed according to Setlow and Carrier (1966).
Survival experiments with near-UV in the presence of psoralens The cell suspensions prepared as described above were exposed to UV around 350 nm (nearUV) at a distance of 10.5 cm from a Toshiba 20-W 'Black-light' lamp in the presence of 8-MOP or angelicin at concentrations of 40 g g / m l or 50 gg/ml, respectively, at room temperature. The dose rate was 25 j/m2/sec, estimated using a near-UV-intensity meter (UVR365, Topcon). The survival estimation was performed as described above.
Sedimentation in alkaline sucrose gradient Cells were labeled with [3H]thymidine as described above. Cell lysis and sedimentation in alkaline sucrose gradient were performed by essentially the same method as previously described (Okubo et al., 1971).
Host-cell reactivation The parental 4698 strain was used in preparation of a fresh lysate of N6 phage (Okubo et al., 1967). N6 phages in PBS were exposed to UV (254 nm) and then infected to M. luteus cells as previously described (Naylor and Burgi, 1956; Okubo et al., 1967). After incubation at 34°C for about 24 h, the number of plaques was counted to estimate phage survival. Preparation and assay of UV endonuclease The ceils were grown at 34°C in LB broth to stationary phase. The cells were harvested by centrifugation, washed twice and resuspended in an original volume of 10 mM Tris-HC1 buffer (pH 7.4) containing 10% sucrose. Cell extracts (fraction II) were prepared according to Carrier and Setlow (1970). Protein was assayed by the method of Lowry et al. (1951). The standard reaction mixture (30 gl) contained 10 mM Tris-HC1 buffer (pH 7.4), 40 mM NaC1, 1 mM flmercaptoethanol, 1 gg of UV-irradiated ColE1 DNA and cell extracts at various concentrations. Incubation was at 37 °C for 20 min. 20/~1 of the reaction mixture was applied to agarose gels (1.0%). Electrophoresis was performed at 40 V for about 4 h. Measurement of thymine dimers Cells were labeled with [3H]thymidine by incubating in nutrient broth supplemented with 5 g C i / m l of [3H]thymidine and 250 g g / m l of deoxyadenosine at 34°C to late log phase. Assay of
Results
Sensitivity of UV- and 4-NQO-sensitive mutants of M. luteus to X-rays and various chemical mutagens UV-sensitive and 4-NQO-sensitive mutants of M. luteus were isolated separately by treatment of the wild-type ATCC4698 strain with MNNG. The mutation frequencies were of the orders of 10-4-10 -3 . The dose-survival curves after exposure to UV and 4-NQO of several representative mutant strains are shown in Fig. 1. The mutant U28 was selected as UV-sensitive mutant and Q57-1 and Q15-2 as 4-NQO-sensitive ones. All of the mutants tested showed increased sensitivity to both UV (Fig. la) and 4-NQO (Fig. lb) in various degrees, compared with the parental strain. The G7 strain was more sensitive to UV than any mutants isolated. It was also found that the UV- and 4-NQOsensitive mutants were highly sensitive to killing by mitomycin C (Fig. 2a) and cis-platinum (Fig. 2b). Furthermore, they were also found to be highly sensitive to near-UV in the presence of bifunctional 8-MOP (Fig. 3a) and monofunctional angelicin (Fig. 3b). On the other hand, as shown in Fig. 4, the mutant, Q57-1, was resistant to X-rays, whereas Q15-2 showed increased sensitivity to X-rays. Host-cell reactivation for UV-damaged N6 phage Fig. 5 shows the survival of UV-irradiated N6 phage when assayed on different host strains. The mutants U28 and Q57-1 were not able to reactivate UV-irradiated N6 phage, but Q15-2 was capable of reactivating the phage. The G7 strain did not exhibit the host-cell reactivation ability, as previously described by Zherebtsov and Tomilin (1982).
234 a
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Fig. 1. Survival curves for stationary phase cultures of various strains of M. luteus after UV irradiation (a) and after 1 h treatment with 4-NQO (b). o, ATCC4698 (parental strain); m, U28; A, Q57-1; v, Q 1 5 - 2 ; . , G7.
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Fig. 2. Survival curves for stationary phase cultures of various strains of M. luteus after 1 h treatment with mitomycin C (a) and cis-platinum (b). Symbols are the same as those for Fig. 1.
235
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Fig. 3. Survival curves for stationary phase cultures of various strains of M. luteus after exposure to near-UV in the presence of 8-MOP (a) and angelicin (b). The concentration of 8-MOP and angelicin were 40 ~g/ml and 50/~g/ml, respectively. Symbols are the same as those for Fig. 1.
U V endonuclease activity in extract of the mutants UV endonuclease activity in the extract was measured by the conversion of UV-irradiated closed-circular form of ColE1 D N A to open-circular form using agarose gel electrophoresis. The results are represented in Fig. 6. There was an endonuclease activity specific for UV-irradiated D N A in the extract of the parental ATCC4698 strain, but it was absent in that of (37. It was evident that the mutants isolated had the normal UV endonuclease activity. In vivo incision of DNA in the parental and mutant strains after exposure to near-UV with angelicin To test the possibility that no incision occurs in the h c r - mutants after exposure to chemical mutagens, the change in the size of D N A in those exposed to near-UV in the presence of angelicin was measured by alkaline sucrose gradient sedimentation after 15 min incubation. The typical
results of D N A sedimentation analysis are shown in Fig. 7. In the parental strain, the molecular size of DNA decreased in the 15 min after near-UV irradiation in the presence of angelicin. However, in the mutant U28, there was no decrease in the size of DNA, indicating that no incision in UVirradiated DNA occurred in the mutant. The in vivo incision could not be detected even when the cells were incubated for 30 min after the treatment. It was also found that the G7 strain could not incise the DNA, as well as the h e r - mutant Q57-1 (data not shown). On the other hand, Q15-2 was able to incise the D N A having angelicin adducts in vivo. Incision of DNA and removal of thymine dimers from DNA in UV-irradiated hcr mutants The removal of thymine dimers from D N A did occur in h e r - mutants at the same rate as in the parental strain, but not in the G7 strain. G7 could -
236
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Fig. 5. UV survival curves for bacteriophage N6 plated on UVand 4-NQO-sensitive m u t a n t s and on parental strain ATCC4698 of M. luteus. Symbols are the same as those for Fig. 1.
Fig. 6. Endonuclease activity in the extract of various strains of M. luteus. ColE1 D N A was irradiated with 200 J / m 2 and then incubated with the extract (fraction II) of M. luteus at 37 ° C for 20 min. All samples were loaded onto a 1% agarose gel (0.04 M Tris acetate-0.1 m M EDTA, p H 8.0), electrophoresed for about 4 h, and then stained with 0 . 5 / ~ g / m l of ethidium bromide. Oc and ccc indicate the mobilities of open circular form and covalently-closed circular form of ColE] D N A , respectively. (a) lanes, a, b, d, f and h, unirradiated D N A ; lanes c, e, g and i, UV-irradiated D N A ; lane a, no enzyme; lanes b - c , d - e , f-g, and h - i , enzyme from ATCC4698, G7, Q57-1, and Q15-2, respectively. 30/~g protein in all cases. (b) lanes a - d and h, unirradiated D N A ; lanes e - g and i-l, UV-irradiated D N A ; lanes a and 1, no enzyme; lanes b - g , enzyme from ATCC4698; lanes h - k , enzyme from U28. Protein contents were 5 / t g (b, e, i), 10/~g (c, f, j) and 21/~g (d, g, h, k).
237
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F r a c t i o n number Fig. 7. Sedimentation patterns in alkaline sucrose-gradient centrifugation of DNA of M. luteus 15 min after treatment with near-UV plus angelicin. The cells were treated with near-UV (10 k J / m 2) plus angelicin (50/xg/ml) and then incubated at 34°C for 15 min. (a) ATCC4698, (b) U28. 0 , immediately after treatment; o, 15 min after treatment.
not incise DNA in the 5 rain after UV irradiation, but, in the parental strain and mutants U28 and Q57-1, the DNA was normally incised in the 5 rain after UV irradiation (data not shown). Discussion
A major mechanism for the repair of DNA damage induced by UV is excision repair. The repair scheme is initiated either by an endonucleolytic cleavage of a phosphodiester bond at or near the site of DNA damage or by hydrolysis of the glycosylic bond between altered base and its sugar moiety (Hanawalt et al., 1979; Lindahl, 1982; Haseltine, 1983). The latter is followed by phosphodiester bond cleavage at the resulting baseless site (Lindahl, 1982). M. luteus possesses a DNA glycosylase activity specific for UV-induced pyrimidine dimers in DNA. Belle and Linn (1982) found that the M. luteus UV endonuclease is actually involved in the in viva excision repair of pyrimidine dimers. However, the question arises as to what degree the UV endonuclease is involved in the resistance of M. luteus to UV. Furthermore, the mechanism for the repair of DNA damage
induced by chemical mutagens such as 4-NQO and psoralen plus near-UV is not clarified. To solve the problems, we have isolated and characterized M. luteus mutants that are highly sensitive to both UV and 4-NQO. The data referring to the repair abilities and sensitivities to chemical mutagens of diffex,ent M. luteus strains are represented in Table 1. It is evident that the mutants possess the UV endonuclease activity (Fig. 6). Furthermore, they are also highly sensitive to mitomycin C, cis-platinum, 8-MOP plus near-UV and angelicin plus near-UV (Figs. 2 and 3). These chemicals induce different types of damage to cellular DNA commonly repaired by the uvr + gene products (UVRABC nuclease) in E. coli (Hanawalt et al., 1979; Sancar and Rupp, 1983). The present experiments indicate that UV-sensitive mutants isolated are defective in the initial step, incision, of the excision-repair pathway operating on bulky DNA damages, because they could not incise the DNA after the treatment of angelicin plus near-UV (Fig. 7). From these results it is concluded that M. luteus possesses an incision mechanism which has a wide substrate specificity like a UVRABC nuclease of E. coli. In addition, it
238 TABLE 1 SOME PROPERTIES OF UV- AND 4NQO-SENSITIVE M. luteus STRAINS IN COMPARISON WITH WILD-TYPE ATCC4698 Strain
ATCC4698 U28 Q57-1 Q15-2 G7
Sensitivity to UV
Bulky adductinducing chemical mutagens
X-Rays
R S S S S
R S S S S
R R R S S
In vivo incision capacity for DNA-containing bulky adducts
Hcr capacity
In vivo incision capacity for UV-irradiated DNA
Removal of thymine dimers
UV endonuclease activity
+
+
+
+
+
_
_
+
+
+
_
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+
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+
+
+
+
+
+
is certain that this r e p a i r p a t h w a y p l a y s an essential role in the resistance of M . luteus to U V a n d various c h e m i c a l mutagens. T h y m i n e d i m e r s are n o r m a l l y r e p a i r e d in U V i r r a d i a t e d hcr m u t a n t strains. I n the m u t a n t s , the incision of D N A is p r o b a b l y p e r f o r m e d b y the U V endonuclease, b e c a u s e the e n z y m a t i c activity is n o t affected b y the m u t a t i o n l e a d i n g to inc r e a s e d sensitivity to U V a n d chemical mutagens. T h e a r g u m e n t is s u p p o r t e d b y the fact that the G 7 strain, defective in the U V e n d o n u c l e a s e ( O k u b o et al., 1971; R i a z u d d i n et al., 1977), c a n n o t incise the U V - i r r a d i a t e d D N A a n d r e m o v e t h y m i n e - c o n t a i n i n g dimers. It is certain t h a t the strain G 7 is defective in b o t h U V e n d o n u c l e a s e activity a n d a n incision m e c h a n i s m o p e r a t i n g b u l k y d a m a g e to D N A , b e c a u s e the m u t a n t is also highly sensitive to various c h e m i c a l m u t a g e n s used (Figs. 2 a n d 3). It is well e s t a b l i s h e d that p y r i m i d i n e d i m e r s p r o d u c e d in D N A u p o n e x p o s u r e to U V are a m a j o r lethal lesion in various o r g a n i s m s ( H a n a walt et al., 1979; F r a n k l i n a n d Haseltine, 1986). M u t a n t s of E. coli that c a n n o t r e p a i r d i m e r s are highly sensitive to killing b y UV. O n the o t h e r h a n d , in the m u t a n t s of M . luteus which show increased sensitivity to U V c o m p a r e d with the p a r e n t a l strain, t h y m i n e d i m e r s are n o r m a l l y removed. These results suggest that t h y m i n e d i m e r s are n o t necessarily the p r i n c i p a l lesion causing cell i n a c t i v a t i o n i n M . luteus. P y r i m i d i n e ( 6 - 4 ) p r o d ucts m a y b e a m a j o r lethal lesion in M . luteus. I d e n t i f i c a t i o n of p o s s i b l e lethal lesions in U V i r r a d i a t e d M . l u t e u s is u n d e r investigation in o u r laboratory.
Acknowledgments T h e a u t h o r s wish to express their g r a t i t u d e to Dr. H. N a k a y a m a , K y u s h u University, for k i n d l y s u p p l y i n g M . luteus strains a n d N 6 p h a g e a n d also for helpful discussions. This w o r k was p a r t l y s u p p o r t e d b y a G r a n t - i n - A i d for Scientific Research (to S.Y.) f r o m the M i n i s t r y of E d u c a t i o n , Science a n d C u l t u r e of Japan.
References Alazard, R., M. Gerrnanier and N.P. Johnson (1982) Mechanisrn of toxicity of platinum(II) compounds in repair-deficient strains of Escherichia coli, Mutation Res., 93, 327-337. Belle, M.L., and S. Linn (1982) In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts. Photochem. Photobiol., 36, 319-324. Carrier, W.L., and R.B. Setlow (1970) Endonuclease from Micrococcus luteus which has activity toward ultravioletirradiated deoxyribonucleic acid: purification and properties, J. Bacteriol., 102, 178-186. Cole, R.S., D. Levitan and R. Sinden (1976) Removal of psoralen interstrand cross-links from DNA of Escherichia coli: Mechanism and genetic control, J. Mol. Biol., 103, 39-59. Franklin, W.A., and W.A. Haseltine (1984) Removal of UV light-induced pyrimidine-pyrimidone(6-4) products from Escherichia coli DNA requires the uvrA, uvrB, and uvrC gene products, Proc. Natl. Acad. Sci. (U.S.A.), 81, 3821-3824. Franklin, W.A., and W.A. Haseltine (1986) The role of the (6-4) photoproducts in ultraviolet light-induced transition mutations in E. coli, Mutation Res., 165, 1-7. Fraval, H.N.A., C.J. Rawlings and J.J. Roberts (1978) Increased sensitivity of UV-repair-deficient human cells to DNA bound platinum products which unlike thymine dimers are not recognized by an endonuclease extracted from Micrococcus luteus, Mutation Res., 51, 121-132.
239 Gordon, L.K., and W.A. Haseltine (1980) Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases, J. Biol. Chem., 255, 12047-12050. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Haseltine, W.A. (1983) Ultraviolet hght repair and mutagenesis revisited, Cell, 33, 13-17. Haseltine, W.A., L.K. Gordon, C.P. Lindan, R.H. Grafstrom, N.L. Shaper and L. Grossman (1980) Cleavage of pyrimidine dimers in specific DNA sequences by a pyrimidine dimer DNA-glycosylase of M. luteus, Nature (London), 285, 634-641. Ikenaga, M., I. Ichikawa-Ryo and S. Kondo (1975) The major cause of inactivation and mutation by 4-nitroquinoline-1oxide in Escherichia coli: Excisable 4NQO-purine adducts, J. Mol. Biol., 92, 341-356. Kondo, S., H. Ichikawa, K. Iwo and T. Kato (1970) Base-change mutagenesis and prophage induction in strains of Escherichia coli with different DNA repair capacities, Genetics, 66, 187-217. Lindahl, T. (1982) DNA repair enzymes, Annu. Rev. Biochem., 51, 61-87. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Fohn phenol reagent, J. Biol. Chem., 193, 265-275. Mahler, I., S.R. Kushner and L. Grossman (1971) In vivo role of the UV-endonuclease from Micrococcus luteus in the repair of DNA, Nature (London), New Biol., 234, 47-50. Nakayama, H., S. Okubo and Y. Takagi (1971) Repair of ultraviolet-damaged DNA in Micrococcus lysodeikticus, I. An endonuclease specific for ultraviolet-irradiated DNA, Biochim. Biophys. Acta, 228, 67-82. Naylor, H.B., and E. Burgi (1956) Observations on abortive infection of Micrococcus lysodeikticus with bacteriophage, Virology, 2, 577-593. Okubo, S., H. Nakayama, M. Sekiguchi and Y. Takagi (1967)
A mutant of Micrococcus lysodeikncus defective in a deoxyribonuclease activity specific for ultraviolet-irradiated DNA, Biochem. Biophys. Res. Commun., 27, 224-229. Okubo, S., H. Nakayama and Y. Takagi (1971) Repair of ultraviolet-damaged DNA in Micrococcus lysodeikticus, II. In vivo investigation on endonuclease activity specific for ultraviolet-irradiated DNA, Biochim. Biophys. Acta, 228, 83-94. Riazuddin, S., and L. Grossman (1977) Micrococcus luteus correndonucleases, I. Resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers, J. Biol. Chem., 252, 6280-6286. Riazuddin, S., L. Grossman and I. Mahler (1977) Micrococcus luteus correndonucleases, III. Evidence for involvement in repair in vivo of two endonucleases specific for DNA containing pyrimidine dimers, J. Biol. Chem., 252, 6294-6298. Sancar, A., and W.D. Rupp (1983) A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33, 249-260. Sancar, A., K.A. Franklin and G. Sancar (1985) Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease, J. Mol. Biol., 184, 725-734. Setlow, R.B., and W.L. Carrier (1966) Pyrimidine dimers in ultraviolet-irradiated DNA's, J. Mol. Biol., 17, 237-254. Takagi, Y., M. Sekiguchi, S. Okubo, H. Nakayama, K. Shimada, S. Yasuda, T. Nishimoto and H. Yoshihara (1968) Nucleases specific for ultraviolet hght-irradiated DNA and their possible role in dark repair, Cold Spring Harbor Symp. Quant. Biol., 33, 219-227. Yeung, A.T., W.B. Mattes, E.Y. Oh and L. Grossman (1983) Enzymatic properties of purified Escherichia coli uvrABC proteins, Proc. Natl. Acad. Sci. (U.S.A.), 80, 6157-6161. Zherebtsov, S.V., and N.V. Tomilin (1982) The roles of different repair mechanisms in the ultraviolet resistance of Micrococcus luteus, Biochim. Biophys. Acta, 698, 295-302.
Mutation Research, 183 (1987) 231-239 DNA Repair Reports Elsevier MTR 06218
The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens Kazuyuki Tao, Asao Noda * and Shuji Yonei Laboratory of Radiation Biology. Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606 (Japan) (Received 25 October 1986) (Revision received 16 December 1986) (Accepted 17 December 1986)
Keywords: Micrococcus luteus mutants; Excision-repair mechanisms; 4-Nitroquinoline 1-oxide; Mitomycin C; Cis-platinum; 8Methoxypsoralen, Angelicin; Endonuclease activity.
Summary M. luteus mutants showing increased sensitivity to both UV and 4-NQO were isolated after the treatment of parental ATCC4698 strain with MNNG. The mutants were also highly sensitive to mitomycin C, cis-platinum, 8-methoxypsoralen (8-MOP) plus near-UV and angelicin plus near-UV in various degrees. The endonuclease activity specific for pyrimidine dimers in UV-irradiated DNA was normally detected in extract of the mutants. With regard to host-cell reactivation ability the mutants fell into two groups. The hcr- mutants lacked the ability to reactivate UV-damaged N6 phage and were resistant to X-rays. The incision of DNA did not occur during incubation after the treatment with angelicin plus near-UV in the hcr- mutants, whereas it occurred in the parental strain. The facts indicate that the hcr- mutants are defective in the incision mechanism which has a wide substrate specificity, similar to the UVRABC nuclease of E. coli. On the other hand, the incision of DNA and the removal of UV-induced thymine dimers from DNA occurred in the hcr- mutants as well as in the parental strain, which is ascribed to the UV endonuclease activity. Compared with the hcr- mutants, hcr ÷ mutants were highly sensitive to X-rays, like r e c A - mutants of E. coli.
The major photoproducts produced in DNA upon exposure to UV are the cyclobutane dimers and the (6-4) photoproducts formed at adjacent pyrimidine bases (Haseltine, 1983). In E. coli, the photoproducts are recognized by a complex of the products of three uvr + genes (uvrA +, uvrB + and uorC +) (Sancar and Rupp, 1983; Yeung et al., * Present address: Meiji Institute of Health Science, 540, Narita, Odawara 250 (Japan). Correspondence: Dr. Shuji Yonei, Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606 (Japan).
1983; Franklin and Haseltine, 1984). The UVRABC nuclease can also recognize DNA damage induced by 4-NQO, bifunctional psoralen plus near-UV, mitomycin C, cis-platinum and monofunctional angelicin plus near-UV (Ikenaga et al., 1975; Cole et al., 1976; Alazard et al., 1982; Sancar and Rupp, 1983; Sancar et al., 1985). Mutants of E. coli defective in one of the uor + genes show increased sensitivity to these agents (Kondo et al., 1970; Hanawalt et al., 1979). The enzymatic activity specific for UV-irradiated DNA has been purified from M. luteus (Carrier and Setlow, 1970; Nakayama et al., 1971;
0167-8817/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
232 Riazuddin and Grossman, 1977). A detailed characterization of the mechanism of action of the M. luteus endonuclease has shown that it cleaves the N-glycosylic bond between the 5'-pyrimidine of a dimer and deoxyribose moiety and then cleaves the phosphodiester bond on the 3'-side of the resulting baseless deoxyribose residues (Haseltine et al., 1980; Gordon and Haseltine, 1980). The M. luteus UV endonuclease is specific for pyrimidine dimers in UV-irradiated DNA (Haseltine et al., 1980). The UV endonuclease has been shown to be involed in in vivo excision repair of pyrimidine dimers in M. luteus (Belle and Linn, 1982). However, a mutant of M. luteus defective in the UV endonuclease activity showed almost the same sensitivity to UV as the wild-type strain (Mahler et al., 1971; Takagi et al., 1968; Riazuddin et al., 1977). On the other hand, a mutant of M. luteus which shows increased sensitivity to UV has normal level of the UV endonuclease activity (Mahler et al., 1971; Zherebtsov and Tomilin, 1982). Thus, the level of endonuclease activity specific for pyrimidine dimers seems not to correlate with the sensitivity of M. luteus to UV. Furthermore, the UV endonuclease cannot recognize D N A damages induced by cis-platinum and psoralen plus nearUV (Fraval et al., 1978; Sancar and Rupp. 1983). From the facts it is possible that M. luteus has another type of incision mechanism which has a wide substrate specificity like the E. coli UVRABC nuclease in addition to the UV endonuclease. If this is the case, a mutant which cannot repair bulky D N A damage induced by UV and various chemical mutagens would be isolated. To test the possibility, we attempted to isolate mutants which show increased sensitivity to both UV and 4-NQO. This paper shows that M. luteus has two different incision mechanisms responsible for DNA damage. Materials and methods
Bacteria and phage The parental ATCC4698 strain and a UV-sensitive strain G7 (Riazuddin et al., 1977) of M. luteus and N6 phage used in this study were kindly supplied by Dr. H. Nakayama, Kyushu University. The ceils were grown at 3 4 ° C in LB broth (10 g bactotryptone [Difco], 5 g yeast extract
[Difco], 5 g NaCI and 1 g glucose in 1000 ml of distilled water, pH 7.2).
Chemicals cis-Diamminedichloroplatinum(II) ( cis-platinum) was a generous gift from Dr. M. Ikenaga, Kyoto University. 4-Nitroquinoline 1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) and mitomycin C were purchased from Wako Pure Chemicals (Osaka). 8-Methoxypsoralen (8-MOP) and angelicin were obtained from Polyscience Inc. (U.S.A.). Colicine E1 (ColE1) D N A was the product of Takara Shuzo (Kyoto). [6-3H]Thymidine was obtained from New England Nuclear (U.S.A.). Isolation of mutants Mutants were isolated after the treatment of parental ATCC4698 strain in stationary phase with 30 / , g / m l of M N N G at 30°C for 30 min. The treated cells were washed twice, resuspended in the original volume of LB broth and then shaken at 34°C for about 15 h. The cells were plated on LB agar and incubated at 34°C for 3 days. UV (250 J/mZ)-sensitive mutants and 4-NQO (100 /,g/ml)-sensitive mutants were isolated separately by replica plating method. Survival experiments with radiation The cells in stationary phase were washed, resuspended in phosphate-buffered saline (PBS, pH 6.5) and then irradiated with UV from germicidal lamps (15-W × 2) at a dose rate of 2 J/mZ/sec, estimated using a UV intensity meter (UVR254, Topcon). X-Irradiation was performed with a Toshiba X-ray machine (KXL-19-2) operating at 180 KVp and 20 mA. The dose rate was 400 r a d / m i n which was measured by the Fricke dosimeter. Following irradiation, the cell suspensions were diluted appropriately and plated on LB agar (2% agar). After incubation at 34°C for about 3 days viable colonies were counted to estimate the survival. Survival experiments with chemicals The cells in stationary phase were washed, resuspended in PBS. The suspensions were kept in the dark at 3 0 ° C for 1 h after the addition of
233 mutagens at various concentrations. The survival estimation was performed as described above.
thymine-containing dimers was performed according to Setlow and Carrier (1966).
Survival experiments with near-UV in the presence of psoralens The cell suspensions prepared as described above were exposed to UV around 350 nm (nearUV) at a distance of 10.5 cm from a Toshiba 20-W 'Black-light' lamp in the presence of 8-MOP or angelicin at concentrations of 40 g g / m l or 50 gg/ml, respectively, at room temperature. The dose rate was 25 j/m2/sec, estimated using a near-UV-intensity meter (UVR365, Topcon). The survival estimation was performed as described above.
Sedimentation in alkaline sucrose gradient Cells were labeled with [3H]thymidine as described above. Cell lysis and sedimentation in alkaline sucrose gradient were performed by essentially the same method as previously described (Okubo et al., 1971).
Host-cell reactivation The parental 4698 strain was used in preparation of a fresh lysate of N6 phage (Okubo et al., 1967). N6 phages in PBS were exposed to UV (254 nm) and then infected to M. luteus cells as previously described (Naylor and Burgi, 1956; Okubo et al., 1967). After incubation at 34°C for about 24 h, the number of plaques was counted to estimate phage survival. Preparation and assay of UV endonuclease The ceils were grown at 34°C in LB broth to stationary phase. The cells were harvested by centrifugation, washed twice and resuspended in an original volume of 10 mM Tris-HC1 buffer (pH 7.4) containing 10% sucrose. Cell extracts (fraction II) were prepared according to Carrier and Setlow (1970). Protein was assayed by the method of Lowry et al. (1951). The standard reaction mixture (30 gl) contained 10 mM Tris-HC1 buffer (pH 7.4), 40 mM NaC1, 1 mM flmercaptoethanol, 1 gg of UV-irradiated ColE1 DNA and cell extracts at various concentrations. Incubation was at 37 °C for 20 min. 20/~1 of the reaction mixture was applied to agarose gels (1.0%). Electrophoresis was performed at 40 V for about 4 h. Measurement of thymine dimers Cells were labeled with [3H]thymidine by incubating in nutrient broth supplemented with 5 g C i / m l of [3H]thymidine and 250 g g / m l of deoxyadenosine at 34°C to late log phase. Assay of
Results
Sensitivity of UV- and 4-NQO-sensitive mutants of M. luteus to X-rays and various chemical mutagens UV-sensitive and 4-NQO-sensitive mutants of M. luteus were isolated separately by treatment of the wild-type ATCC4698 strain with MNNG. The mutation frequencies were of the orders of 10-4-10 -3 . The dose-survival curves after exposure to UV and 4-NQO of several representative mutant strains are shown in Fig. 1. The mutant U28 was selected as UV-sensitive mutant and Q57-1 and Q15-2 as 4-NQO-sensitive ones. All of the mutants tested showed increased sensitivity to both UV (Fig. la) and 4-NQO (Fig. lb) in various degrees, compared with the parental strain. The G7 strain was more sensitive to UV than any mutants isolated. It was also found that the UV- and 4-NQOsensitive mutants were highly sensitive to killing by mitomycin C (Fig. 2a) and cis-platinum (Fig. 2b). Furthermore, they were also found to be highly sensitive to near-UV in the presence of bifunctional 8-MOP (Fig. 3a) and monofunctional angelicin (Fig. 3b). On the other hand, as shown in Fig. 4, the mutant, Q57-1, was resistant to X-rays, whereas Q15-2 showed increased sensitivity to X-rays. Host-cell reactivation for UV-damaged N6 phage Fig. 5 shows the survival of UV-irradiated N6 phage when assayed on different host strains. The mutants U28 and Q57-1 were not able to reactivate UV-irradiated N6 phage, but Q15-2 was capable of reactivating the phage. The G7 strain did not exhibit the host-cell reactivation ability, as previously described by Zherebtsov and Tomilin (1982).
234 a
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Fig. 1. Survival curves for stationary phase cultures of various strains of M. luteus after UV irradiation (a) and after 1 h treatment with 4-NQO (b). o, ATCC4698 (parental strain); m, U28; A, Q57-1; v, Q 1 5 - 2 ; . , G7.
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Fig. 2. Survival curves for stationary phase cultures of various strains of M. luteus after 1 h treatment with mitomycin C (a) and cis-platinum (b). Symbols are the same as those for Fig. 1.
235
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Fig. 3. Survival curves for stationary phase cultures of various strains of M. luteus after exposure to near-UV in the presence of 8-MOP (a) and angelicin (b). The concentration of 8-MOP and angelicin were 40 ~g/ml and 50/~g/ml, respectively. Symbols are the same as those for Fig. 1.
U V endonuclease activity in extract of the mutants UV endonuclease activity in the extract was measured by the conversion of UV-irradiated closed-circular form of ColE1 D N A to open-circular form using agarose gel electrophoresis. The results are represented in Fig. 6. There was an endonuclease activity specific for UV-irradiated D N A in the extract of the parental ATCC4698 strain, but it was absent in that of (37. It was evident that the mutants isolated had the normal UV endonuclease activity. In vivo incision of DNA in the parental and mutant strains after exposure to near-UV with angelicin To test the possibility that no incision occurs in the h c r - mutants after exposure to chemical mutagens, the change in the size of D N A in those exposed to near-UV in the presence of angelicin was measured by alkaline sucrose gradient sedimentation after 15 min incubation. The typical
results of D N A sedimentation analysis are shown in Fig. 7. In the parental strain, the molecular size of DNA decreased in the 15 min after near-UV irradiation in the presence of angelicin. However, in the mutant U28, there was no decrease in the size of DNA, indicating that no incision in UVirradiated DNA occurred in the mutant. The in vivo incision could not be detected even when the cells were incubated for 30 min after the treatment. It was also found that the G7 strain could not incise the DNA, as well as the h e r - mutant Q57-1 (data not shown). On the other hand, Q15-2 was able to incise the D N A having angelicin adducts in vivo. Incision of DNA and removal of thymine dimers from DNA in UV-irradiated hcr mutants The removal of thymine dimers from D N A did occur in h e r - mutants at the same rate as in the parental strain, but not in the G7 strain. G7 could -
236
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X rays (Krad) Fig. 4. Survival curves of various strains of M. luteus after X-irradiation. Symbols are the same as those for Fig. 1.
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Fig. 5. UV survival curves for bacteriophage N6 plated on UVand 4-NQO-sensitive m u t a n t s and on parental strain ATCC4698 of M. luteus. Symbols are the same as those for Fig. 1.
Fig. 6. Endonuclease activity in the extract of various strains of M. luteus. ColE1 D N A was irradiated with 200 J / m 2 and then incubated with the extract (fraction II) of M. luteus at 37 ° C for 20 min. All samples were loaded onto a 1% agarose gel (0.04 M Tris acetate-0.1 m M EDTA, p H 8.0), electrophoresed for about 4 h, and then stained with 0 . 5 / ~ g / m l of ethidium bromide. Oc and ccc indicate the mobilities of open circular form and covalently-closed circular form of ColE] D N A , respectively. (a) lanes, a, b, d, f and h, unirradiated D N A ; lanes c, e, g and i, UV-irradiated D N A ; lane a, no enzyme; lanes b - c , d - e , f-g, and h - i , enzyme from ATCC4698, G7, Q57-1, and Q15-2, respectively. 30/~g protein in all cases. (b) lanes a - d and h, unirradiated D N A ; lanes e - g and i-l, UV-irradiated D N A ; lanes a and 1, no enzyme; lanes b - g , enzyme from ATCC4698; lanes h - k , enzyme from U28. Protein contents were 5 / t g (b, e, i), 10/~g (c, f, j) and 21/~g (d, g, h, k).
237
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F r a c t i o n number Fig. 7. Sedimentation patterns in alkaline sucrose-gradient centrifugation of DNA of M. luteus 15 min after treatment with near-UV plus angelicin. The cells were treated with near-UV (10 k J / m 2) plus angelicin (50/xg/ml) and then incubated at 34°C for 15 min. (a) ATCC4698, (b) U28. 0 , immediately after treatment; o, 15 min after treatment.
not incise DNA in the 5 rain after UV irradiation, but, in the parental strain and mutants U28 and Q57-1, the DNA was normally incised in the 5 rain after UV irradiation (data not shown). Discussion
A major mechanism for the repair of DNA damage induced by UV is excision repair. The repair scheme is initiated either by an endonucleolytic cleavage of a phosphodiester bond at or near the site of DNA damage or by hydrolysis of the glycosylic bond between altered base and its sugar moiety (Hanawalt et al., 1979; Lindahl, 1982; Haseltine, 1983). The latter is followed by phosphodiester bond cleavage at the resulting baseless site (Lindahl, 1982). M. luteus possesses a DNA glycosylase activity specific for UV-induced pyrimidine dimers in DNA. Belle and Linn (1982) found that the M. luteus UV endonuclease is actually involved in the in viva excision repair of pyrimidine dimers. However, the question arises as to what degree the UV endonuclease is involved in the resistance of M. luteus to UV. Furthermore, the mechanism for the repair of DNA damage
induced by chemical mutagens such as 4-NQO and psoralen plus near-UV is not clarified. To solve the problems, we have isolated and characterized M. luteus mutants that are highly sensitive to both UV and 4-NQO. The data referring to the repair abilities and sensitivities to chemical mutagens of diffex,ent M. luteus strains are represented in Table 1. It is evident that the mutants possess the UV endonuclease activity (Fig. 6). Furthermore, they are also highly sensitive to mitomycin C, cis-platinum, 8-MOP plus near-UV and angelicin plus near-UV (Figs. 2 and 3). These chemicals induce different types of damage to cellular DNA commonly repaired by the uvr + gene products (UVRABC nuclease) in E. coli (Hanawalt et al., 1979; Sancar and Rupp, 1983). The present experiments indicate that UV-sensitive mutants isolated are defective in the initial step, incision, of the excision-repair pathway operating on bulky DNA damages, because they could not incise the DNA after the treatment of angelicin plus near-UV (Fig. 7). From these results it is concluded that M. luteus possesses an incision mechanism which has a wide substrate specificity like a UVRABC nuclease of E. coli. In addition, it
238 TABLE 1 SOME PROPERTIES OF UV- AND 4NQO-SENSITIVE M. luteus STRAINS IN COMPARISON WITH WILD-TYPE ATCC4698 Strain
ATCC4698 U28 Q57-1 Q15-2 G7
Sensitivity to UV
Bulky adductinducing chemical mutagens
X-Rays
R S S S S
R S S S S
R R R S S
In vivo incision capacity for DNA-containing bulky adducts
Hcr capacity
In vivo incision capacity for UV-irradiated DNA
Removal of thymine dimers
UV endonuclease activity
+
+
+
+
+
_
_
+
+
+
_
_
+
+
+
+
+
+
+
+
is certain that this r e p a i r p a t h w a y p l a y s an essential role in the resistance of M . luteus to U V a n d various c h e m i c a l mutagens. T h y m i n e d i m e r s are n o r m a l l y r e p a i r e d in U V i r r a d i a t e d hcr m u t a n t strains. I n the m u t a n t s , the incision of D N A is p r o b a b l y p e r f o r m e d b y the U V endonuclease, b e c a u s e the e n z y m a t i c activity is n o t affected b y the m u t a t i o n l e a d i n g to inc r e a s e d sensitivity to U V a n d chemical mutagens. T h e a r g u m e n t is s u p p o r t e d b y the fact that the G 7 strain, defective in the U V e n d o n u c l e a s e ( O k u b o et al., 1971; R i a z u d d i n et al., 1977), c a n n o t incise the U V - i r r a d i a t e d D N A a n d r e m o v e t h y m i n e - c o n t a i n i n g dimers. It is certain t h a t the strain G 7 is defective in b o t h U V e n d o n u c l e a s e activity a n d a n incision m e c h a n i s m o p e r a t i n g b u l k y d a m a g e to D N A , b e c a u s e the m u t a n t is also highly sensitive to various c h e m i c a l m u t a g e n s used (Figs. 2 a n d 3). It is well e s t a b l i s h e d that p y r i m i d i n e d i m e r s p r o d u c e d in D N A u p o n e x p o s u r e to U V are a m a j o r lethal lesion in various o r g a n i s m s ( H a n a walt et al., 1979; F r a n k l i n a n d Haseltine, 1986). M u t a n t s of E. coli that c a n n o t r e p a i r d i m e r s are highly sensitive to killing b y UV. O n the o t h e r h a n d , in the m u t a n t s of M . luteus which show increased sensitivity to U V c o m p a r e d with the p a r e n t a l strain, t h y m i n e d i m e r s are n o r m a l l y removed. These results suggest that t h y m i n e d i m e r s are n o t necessarily the p r i n c i p a l lesion causing cell i n a c t i v a t i o n i n M . luteus. P y r i m i d i n e ( 6 - 4 ) p r o d ucts m a y b e a m a j o r lethal lesion in M . luteus. I d e n t i f i c a t i o n of p o s s i b l e lethal lesions in U V i r r a d i a t e d M . l u t e u s is u n d e r investigation in o u r laboratory.
Acknowledgments T h e a u t h o r s wish to express their g r a t i t u d e to Dr. H. N a k a y a m a , K y u s h u University, for k i n d l y s u p p l y i n g M . luteus strains a n d N 6 p h a g e a n d also for helpful discussions. This w o r k was p a r t l y s u p p o r t e d b y a G r a n t - i n - A i d for Scientific Research (to S.Y.) f r o m the M i n i s t r y of E d u c a t i o n , Science a n d C u l t u r e of Japan.
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239 Gordon, L.K., and W.A. Haseltine (1980) Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases, J. Biol. Chem., 255, 12047-12050. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Haseltine, W.A. (1983) Ultraviolet hght repair and mutagenesis revisited, Cell, 33, 13-17. Haseltine, W.A., L.K. Gordon, C.P. Lindan, R.H. Grafstrom, N.L. Shaper and L. Grossman (1980) Cleavage of pyrimidine dimers in specific DNA sequences by a pyrimidine dimer DNA-glycosylase of M. luteus, Nature (London), 285, 634-641. Ikenaga, M., I. Ichikawa-Ryo and S. Kondo (1975) The major cause of inactivation and mutation by 4-nitroquinoline-1oxide in Escherichia coli: Excisable 4NQO-purine adducts, J. Mol. Biol., 92, 341-356. Kondo, S., H. Ichikawa, K. Iwo and T. Kato (1970) Base-change mutagenesis and prophage induction in strains of Escherichia coli with different DNA repair capacities, Genetics, 66, 187-217. Lindahl, T. (1982) DNA repair enzymes, Annu. Rev. Biochem., 51, 61-87. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Fohn phenol reagent, J. Biol. Chem., 193, 265-275. Mahler, I., S.R. Kushner and L. Grossman (1971) In vivo role of the UV-endonuclease from Micrococcus luteus in the repair of DNA, Nature (London), New Biol., 234, 47-50. Nakayama, H., S. Okubo and Y. Takagi (1971) Repair of ultraviolet-damaged DNA in Micrococcus lysodeikticus, I. An endonuclease specific for ultraviolet-irradiated DNA, Biochim. Biophys. Acta, 228, 67-82. Naylor, H.B., and E. Burgi (1956) Observations on abortive infection of Micrococcus lysodeikticus with bacteriophage, Virology, 2, 577-593. Okubo, S., H. Nakayama, M. Sekiguchi and Y. Takagi (1967)
A mutant of Micrococcus lysodeikncus defective in a deoxyribonuclease activity specific for ultraviolet-irradiated DNA, Biochem. Biophys. Res. Commun., 27, 224-229. Okubo, S., H. Nakayama and Y. Takagi (1971) Repair of ultraviolet-damaged DNA in Micrococcus lysodeikticus, II. In vivo investigation on endonuclease activity specific for ultraviolet-irradiated DNA, Biochim. Biophys. Acta, 228, 83-94. Riazuddin, S., and L. Grossman (1977) Micrococcus luteus correndonucleases, I. Resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers, J. Biol. Chem., 252, 6280-6286. Riazuddin, S., L. Grossman and I. Mahler (1977) Micrococcus luteus correndonucleases, III. Evidence for involvement in repair in vivo of two endonucleases specific for DNA containing pyrimidine dimers, J. Biol. Chem., 252, 6294-6298. Sancar, A., and W.D. Rupp (1983) A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33, 249-260. Sancar, A., K.A. Franklin and G. Sancar (1985) Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease, J. Mol. Biol., 184, 725-734. Setlow, R.B., and W.L. Carrier (1966) Pyrimidine dimers in ultraviolet-irradiated DNA's, J. Mol. Biol., 17, 237-254. Takagi, Y., M. Sekiguchi, S. Okubo, H. Nakayama, K. Shimada, S. Yasuda, T. Nishimoto and H. Yoshihara (1968) Nucleases specific for ultraviolet hght-irradiated DNA and their possible role in dark repair, Cold Spring Harbor Symp. Quant. Biol., 33, 219-227. Yeung, A.T., W.B. Mattes, E.Y. Oh and L. Grossman (1983) Enzymatic properties of purified Escherichia coli uvrABC proteins, Proc. Natl. Acad. Sci. (U.S.A.), 80, 6157-6161. Zherebtsov, S.V., and N.V. Tomilin (1982) The roles of different repair mechanisms in the ultraviolet resistance of Micrococcus luteus, Biochim. Biophys. Acta, 698, 295-302.