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UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog
Mutation Research, DNA Repair, 273 (1992)43-48 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00
43
MUTDNA 06469
UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog Hiroaki Nakayama, Susumu Shiota and Keiko Umezu Department of Microbiology, Faculty of Dentistry, Kyushu Unh'ersity, Higashi-ku, Fukuoka 812 (Japan) (Received 7 March 1991) (Accepted 6 June 1991)
Keywords: Excision repair; Micrococcus luteus; UV damage; UV endonuclease
Summary Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonucle. ase (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e.. mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coil UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. "~' ,", ~l results indicate .......... that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.
UV endonuclease, thus far identified only in Micrococcus luteus and phage T4 (also known as cndonuclease V in the latter), is a dual-action enzyme having a pyrimidine dimer DNA glycosylase and an apurinic-apyrimidinic (AP) endonuclease activity (see Friedberg, 1985, for a review). The former activity cleaves the 5'-glycosylic bond of a UV-induced pyrimidine dimer in DNA,
Correspondence: H. Nakayama, Department of Microbiology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka 812 (Japan).
whereas the latter cuts the phosphodiester linkage 3' to the AP site thus formed or an AP site generated otherwise. Apart from its apparently non-specific AP endonuclease activity, UV endonuclease appears to be specific for pyrimidine dimers and, at variance with UvrABC endonuclease of Escherichia coli, to be inactive on DNA containing UV-induced 6-4 photoproducts or base adducts generated by chemicals such as mitomycin C and 4-nitroquinoline 1-oxide. There is biochemical evidence that M. luteus UV endonuclease represents the first line of defence against UV-induced pyrimidine dimers.
44
Once acted upon by this enzyme, a pyrimidine dimer in DNA is connected with the rest of the DNA molecule solely through its 3'-glycosylic bond, and monomerization (reverse reaction) effected by a second UV irradiation is expected to release the 5'-pyrimidine. Taking advantage of this unique feature, LaBell and Linn (1982) were able to show that in wild-type M. luteus cells most thymine-containing pyrimidine dimers excised from DNA actually retain the proof for the involvement of UV endonuclease in the excision. Curiously, however, cell survival data so far reported do not apparently indicate such a major role for UV endonuclease. Thus, most UV endonuclease-deficient derivatives show normal UV sensitivity (Okubo et al., 1971), and, conversely, most UV-sensitive mutants exhibit normal levels of UV endonuclease activity (Okubo et al., 1971). This apparent contradiction led us to show, by gene cloning and sequencing, that M. luteus has genes for a 'backup' enzyme, a homolog of E. coil UvrABC endonuclease (Shiota and Nakayama, 1988, 1989). In view of the broad substrate specificity of the E. coil enzyme, this finding neatly explains the repeated observation of the increased sensitivity to adduct-forming chemicals of certain M. luteus mutants now known or supposed to be defective in this Uvr-type enzyme (Okubo et al., 1971; Riazuddin and Grossman, 1967; Tap et al., 1987). Although the discovery of the M. haeus genes homologous to E. coil uvrA and uvrB accounted for many of the previously puzzling observations (Shiota and Nakayama, 1988, 1989), UV endonuclease was left without positive evidence implicating it in UV survival of the cell. This is in sharp contrast to the situation of phage T4, in which denV (formerly v) mutants (Harm, 1963), lacking endonuclease V, are definitely more UV-sensitive than the wild type, clearly indicating that the T4 enzyme contributes to UV survival of the phage (Friedberg and King, 1969; Sekiguchi et al., 1970). Reasoning that the elimination of the backup by the Uvr system may disclose evidence for the promotion of UV survival of M. haeus cells by UV endonuclease, we carried out killing experiments using Uvr-deficient mutants with or without UV endonuclease activity. Here, we report the results of these studies, which, as expected,
clearly showed the contribution of the enzyme to UV survival of the bacterium. Materials and methods
Bacterial strains and culture conditions M. luteus wild-type strain ATCC4698, its UVsensitive derivatives DB7 (formerly G7 or 7) (Grossman et al., 1968; Shiota and Nakayama, 1989) and UVSNI (Okubo et al., 1971; Shiota and Nakayama, 1988), and a UV-resistant transformant of DB7 (Shiota and Nakayama, 1989) were used. UV"NI is deficient in a gene homologous to uvrB of E. coil (Shiota and Nakayama, 1988), whereas DB7 is defective in UV endonuclease activity (Grossman et al. 1968; Okubo et al., 1971) as well as in the homolog of E. coil uvrA (Shiota and Nakayama, 1989). The transformant is defective only in UV endonuelease (Shiota and Nakayama, 1989). Cells were routinely grown in a mineral-supplemented broth medium (Okubo and Nakayama, 1968) with shaking at 32 o C. Nutrient agar for the survival assay was as described (Nakayama et ai., 1982), and plates were incubated at 32 °C for 2 days. Survival studies with mitomycin C and 4. nitroquinoline /.oxide Cells from log-phase cultures (4-10× l0 T cfu/ml) were washed by low-speed centrifugation with phosphate-buffered saline (PBS, 13.6 mM Na.,HPOJKH2PO 4, pH 7.0, 0.12 M NaCI), and suspended in the original volume of the same buffer. To portions of the suspension were added appropriate amounts of an aqueous solution of each chemical so as to give desired concentrations of the chemical, and the mixtures were incubated at 32 °C for 60 rain. Appropriate dilutions of the mixtures were made in PBS, and samples were spread on nutrient agar plates. Survival studies with 254-nm UV Log-phase cultures were diluted 2000-fold into PBS, and a 5-mi portion of the dilution was irradiated under a 5-W germicidal lamp at a dose rate of 0.12 j / m 2 / s . Samples were taken at inter. vals and spread on nutrient agar plates after dilution with PBS when appropriate.
45 Sun,ival studies with molecular photosensitization Cells were harvested from log-phase cultures by centrifugation and suspended in the original volume of PBS. The suspension was diluted 100fold into a 9: 1 (v/v) mixture of PBS and acetone, and left standing at room temperature for 5 min. It was then illuminated under a 312-rim light source (VL-30M, Vilber Lourmat, Marne la Vall6e, France) at a distance of 10 cm, and samples taken at intervals were spread on nutrient agar plates after dilution with PBS when needed. Acetone did not affect the colony-forming ability of the cells of each strain for at least 20 min at this concentration.
whereas strain DB7 is defective in UV endonuclease activity (Grossman, 1968; Okubo et al., 1971) as well as in the urrA homolog (Shiota and Nakayama, 1989). Hence, comparison of these strains should provide information about the effect of UV endonuclease on the UV survival of the cells as measured in the absence of interference by the Uvr homolog. For this comparison to be valid, however, it is required that both mutant strains be defective in the activity of the Uvr homolog to exactly the same degree. Unfortunately, such activity in wild-type M. luteus has not yet been detected in vitro. We therefore examined both strains for sensitivity to mitomycin C and 4-nitroquinoli,e 1-oxide. These chemicals are known to form adducts in DNA which are subject to repair by the E. coil Uvr system (Van Houten, 1990). Perhaps the M. luteus Uvr system can also repair such damage as judged by the sensitivity of U V ~ : I and DB7 to these chemicals (Shiota and Nakayama, 1988, 1989), whereas UV endonucle-
Results and discussion According to previous work, strain UVSN1 is mutant for the homolog of E. coil ul.'rB (Shiota and Nakayama, 1988) but normal with respect to UV endonuclease activity (Okubo et al., 1971),
i
ia
I Re---.~
^
b •
16 2 I
0
I
I
I
t
0.2
0.4
0.6
0.8
Mitomycin C ( gg ml-~ )
~
i
O
I
I
0.1
0°2
I
I
0.3
0.4
4-Nitroquinoline l-oxide (mM)
Fig. 1. Sensitivityof M. luteus strains to mitomycinC (a) or 4-nitroquinoline l-oxide (b). The results shown are those of typical experiments, o, DB7 (uL'rA. UV endonuclease-deficient);e, UV~NI (uurB, UV endonucleasenormal): zx, ATCC4698(wild type): A, uvr+ transformantof DB7 (UV endonuclease-deficient).
46
ase, which is highly specific for pyrimidine dimers, cannot. Consequently, it was anticipated that if the 2 strains are equivalent in the degree of Uvr deficiency, they should exhibit identical sensitivity to each of these chemicals irrespective of the presence or absence of UV endonuclease. This expectation was in fact realized as shown in Fig. 1. It should be noted that the equal sensitivity was seen with 2 different substances that are unrelated to each other in terms of chemical structure and the mechanism for enzymatic activation (Szybalski and Iyer, 1964, Tada, 1981). If one assumes that the defects of the Uvr homolog are different in degree between the 2 mutants, it must be compensated by a difference in some other factor, e.g., permeation or activation, for the identical sensitivity to result. It is highly unlikely that such compensation would occur with the 2 unrelated substances. We therefore conclude that the 2 strains have Uvr deficiencies
10-'
1(~2
0
60
120 180 Oose (sec)
240
300
Fig. 3. Sensitivity of M, luteus strains to molecular photosensitization, Survival rates are represented by means (symbols) and SDs (vertical bars) for 4 (UV'NI) or 5 (DBT) separate experiments. Abscissa. time of irradiation at 312 nm. o, DB?; e. UV~NI,
,I I
"~D 11~I |m
I f,4
l,
I
0
60
I
120 Dose (sec)
I
180
Fig. 2. Sensitivity of M, luteus strains to 254-nm UV light, Survival rates are represented by means (symbols) and SDs (vertical bars) of 4 separate experiments, Abscissa, time of irradiation, o, DB7; e. UV~NI,
functionally indistinguishable from each other. Perhaps both strains are 'null' mutants in view of the fact that DB7 has a nonsense mutation in the ut'rA homolog (Shiota and Nakayama, 1989). As shown in Fig. 2, DB7 was substantially more sensitive to 254-nm UV than UVSNI, with a dose reduction factor of around 2 at the survival level of 37%. In the light of the above-mentioned results with the chemicals, we considered it likely that this difference in UV survival reflected the presence or absence of UV endonuclease in these mutants. In order to strengthen this interpretation, we went on to look at the effect of molecular photosensitization with acetone as a sensitizer. Unlike 254-nm irradiation, which yields not only pyrimidine dimers but also a significant amount of 6-4 photoproducts, the acetone-mediated molecular photosensitization
47
has been shown to generate exclusively pyrimidine dimers (Ben-lshai, 1968). Consequently, if UV endonuclease is responsible for the differential killing of the 2 strains by 254-nm UV (Fig. 2), it was anticipated that the photosensitization ~'~uld reveal a greater strain difference in sensitivity. The results presented in Fig. 3 indicate that this was actually the case: DB7 was more sensitive to acetone plus 312-nm light than UV~N1, with an increased dose reduction facto( of about 3.4 at the 37% survival level. Incidentally, the wild-type strain and the UV endonuclease-deficient Uvr + transformant did not incur any loss of viability in the dose ranges shown in Figs. 2 and 3. From the above results, it seems highly likely that the difference in the sensitivity to 254-nm UV as well as to the acetone-mediated photosensitization between UV"NI and DB7 reflects the in vivo activity of UV endonuclease. Consistent with this idea is the observation by Tao et al. (1987) that their M. luteus mutants, which are similar to UV"NI, are invariably less UV-sensitire than, but as susceptible to adduct-forming chemicals as, DB7. As has previously been shown (Okubo etai., 1971; Shiota and Nakayama, 1989), a defect in UV endonuclease alone does not produce any appreciable effect on the sensitivity of the cell to 254-nm UV over a wide dose range. Unlike such M. luteus mutants, UV endonuclease (endonuclease V)-deficient (denV) mutants of phage T4 have long been known to be more UV-sensitire than the wild-type (Harm, 1963). Since T4 is insusceptible to host.cell reactivation (Ell(son et al., 1960), UV endonuclease should be the sole incision enzyme for this virus that is responsible for the excision repair of UV damage. Hence I"4 can now be regarded as equivalent to Uvr-deficient M. luteus mutants such as UV~N1, in which the incision reaction should also be dependent solely on UV endonuclease. We consider that the long-standing, apparent discrepancy between T4 and M. luteus with respect to the in vivo role for UV endonuclease has now been solved, with the unified view that UV endonuclease is functional in rive, with its contribution to UV survival being manifest only in the absence of another efficient incision system(s).
Acknowledgements We are grateful to Ms. C. Yoshida for her general supportive services in the laboratory. Technical help provided by Dr. K. Nakayama in an early phase of this work is acknowledged with appreciation.
References Ben-lshai, R., E. Ben-Hur and Y. Horn(old (1968) Photosensitized dimerization of thymine and cytosine in DNA, Israel J. Chem., 6, 769-775. Ell(son, S.A., R.R. Feiner and R.F. lift[ (1960) A host effect on bacteriophage survival after ultraviolet irradiation, Virology, 1I, 294-296. Friedberg, E.C. (1985) DNA Repair, Freeman, New York. Friedberg, E.C., and J.J. King (1969) Endonucleolytic cleavage of UV-irradiated DNA controlled by the v ÷ gene in phage T4, Biochem. Biophys, Res. Commun., 37, 646-651. Grossman, L., J.Co Kaplan, S.R. Kushner and 1. Mahler (1968) Enzymes involved in the early stages of repair of ultraviolet-irradiated DNA, Cold Spring Harbor Syrup. Quant. Biol., 33, 229-234. Harm, W. (1963) Mutants of phage T4 with increased sensitivity to ultraviolet, Virology, 19, 66-71. LaBell, M., and S. Linn 0982) In rive excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococ. cus haeus bul not in human flhroblasts, Photochem. Pholob(el., 36, 319-324. Nakayama, H,, K, Nukayama, R. Nakayama and Y. Nakayama (1982) Recombination-deficient mutations and Ihymineless death in h;~cherichia col( KI2: reciprocal of(eels of recBC and reef and indifference of recA mutations, Can. J. Mierobiol,, 28, 425-430, Okubo, S,, and H. Nakayama (1968) Evidence of transformation in Micrococcus ly.~odeiktic~c.~, Bioehem. Biophys. Res, Commun., 32, 825-830. Okubo, S., H. Nakayama and Y. Takagi (1971) Repair of ultraviolet-damaged DNA in Mtcrococcu.~ lysodeiktic¢~s, II. In vivo investigation on endonuclease activity specific fi)r ultraviolet-irradiated DNA, Biochim. Biophys. Acta, 228, 83 -94. Sancar, A., and G.B. Sancar (1988) DNA repair enzymes. Anna. Roy, Biochem.. 57, 29-67. Shiota, S., and H. Nakayama (1988) Evidence for a MicrococcL~s h~te4cs gone homologous to ~ rB of Escherichia coli, Mol, Gen. Goner., 213, 21-29. Shiota, S., and H. Nakayama (1989) Micrococcus luteus homolog of the Escherichia col( urrA gone: identification of a mutation in the UV-sensitive mutant DB7, Mol. Gen. Goner., 217, 332-340. Szybalski, W., and V.N. lyer (1967) The mitomycins and porfiromycins, in: D. Gottlieb and P.D. Shaw (Eds.). Antibiotics, Vol. I, Mechanism of Action, Springer, Berlin, pp. 21 i-245.
48
Tada, M. (1981) Metabolism of 4-nitroquinoline l-oxide and related compounds, in: T. Sugimura (Ed.), Carcinogenesis, Vol. 6, Raven, New York, pp. 25-45. Tao, K., A. Noda and S. Yonei (1987) The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens, Mutation Res., 183, 231-239.
Van Houten, B. (1990) Nucleotide excision repair in Escherichia coil, Microbiol. Roy., 54, 18-51. Yasuda, S., and M. Sekiguchi (1970) 3"4 endonuclease involved in repair of DNA, Proc. Natl. Acad. Sci. (U.S.A.), 67, 1839-1845.
43
MUTDNA 06469
UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog Hiroaki Nakayama, Susumu Shiota and Keiko Umezu Department of Microbiology, Faculty of Dentistry, Kyushu Unh'ersity, Higashi-ku, Fukuoka 812 (Japan) (Received 7 March 1991) (Accepted 6 June 1991)
Keywords: Excision repair; Micrococcus luteus; UV damage; UV endonuclease
Summary Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonucle. ase (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e.. mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coil UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. "~' ,", ~l results indicate .......... that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.
UV endonuclease, thus far identified only in Micrococcus luteus and phage T4 (also known as cndonuclease V in the latter), is a dual-action enzyme having a pyrimidine dimer DNA glycosylase and an apurinic-apyrimidinic (AP) endonuclease activity (see Friedberg, 1985, for a review). The former activity cleaves the 5'-glycosylic bond of a UV-induced pyrimidine dimer in DNA,
Correspondence: H. Nakayama, Department of Microbiology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka 812 (Japan).
whereas the latter cuts the phosphodiester linkage 3' to the AP site thus formed or an AP site generated otherwise. Apart from its apparently non-specific AP endonuclease activity, UV endonuclease appears to be specific for pyrimidine dimers and, at variance with UvrABC endonuclease of Escherichia coli, to be inactive on DNA containing UV-induced 6-4 photoproducts or base adducts generated by chemicals such as mitomycin C and 4-nitroquinoline 1-oxide. There is biochemical evidence that M. luteus UV endonuclease represents the first line of defence against UV-induced pyrimidine dimers.
44
Once acted upon by this enzyme, a pyrimidine dimer in DNA is connected with the rest of the DNA molecule solely through its 3'-glycosylic bond, and monomerization (reverse reaction) effected by a second UV irradiation is expected to release the 5'-pyrimidine. Taking advantage of this unique feature, LaBell and Linn (1982) were able to show that in wild-type M. luteus cells most thymine-containing pyrimidine dimers excised from DNA actually retain the proof for the involvement of UV endonuclease in the excision. Curiously, however, cell survival data so far reported do not apparently indicate such a major role for UV endonuclease. Thus, most UV endonuclease-deficient derivatives show normal UV sensitivity (Okubo et al., 1971), and, conversely, most UV-sensitive mutants exhibit normal levels of UV endonuclease activity (Okubo et al., 1971). This apparent contradiction led us to show, by gene cloning and sequencing, that M. luteus has genes for a 'backup' enzyme, a homolog of E. coil UvrABC endonuclease (Shiota and Nakayama, 1988, 1989). In view of the broad substrate specificity of the E. coil enzyme, this finding neatly explains the repeated observation of the increased sensitivity to adduct-forming chemicals of certain M. luteus mutants now known or supposed to be defective in this Uvr-type enzyme (Okubo et al., 1971; Riazuddin and Grossman, 1967; Tap et al., 1987). Although the discovery of the M. haeus genes homologous to E. coil uvrA and uvrB accounted for many of the previously puzzling observations (Shiota and Nakayama, 1988, 1989), UV endonuclease was left without positive evidence implicating it in UV survival of the cell. This is in sharp contrast to the situation of phage T4, in which denV (formerly v) mutants (Harm, 1963), lacking endonuclease V, are definitely more UV-sensitive than the wild type, clearly indicating that the T4 enzyme contributes to UV survival of the phage (Friedberg and King, 1969; Sekiguchi et al., 1970). Reasoning that the elimination of the backup by the Uvr system may disclose evidence for the promotion of UV survival of M. haeus cells by UV endonuclease, we carried out killing experiments using Uvr-deficient mutants with or without UV endonuclease activity. Here, we report the results of these studies, which, as expected,
clearly showed the contribution of the enzyme to UV survival of the bacterium. Materials and methods
Bacterial strains and culture conditions M. luteus wild-type strain ATCC4698, its UVsensitive derivatives DB7 (formerly G7 or 7) (Grossman et al., 1968; Shiota and Nakayama, 1989) and UVSNI (Okubo et al., 1971; Shiota and Nakayama, 1988), and a UV-resistant transformant of DB7 (Shiota and Nakayama, 1989) were used. UV"NI is deficient in a gene homologous to uvrB of E. coil (Shiota and Nakayama, 1988), whereas DB7 is defective in UV endonuclease activity (Grossman et al. 1968; Okubo et al., 1971) as well as in the homolog of E. coil uvrA (Shiota and Nakayama, 1989). The transformant is defective only in UV endonuelease (Shiota and Nakayama, 1989). Cells were routinely grown in a mineral-supplemented broth medium (Okubo and Nakayama, 1968) with shaking at 32 o C. Nutrient agar for the survival assay was as described (Nakayama et ai., 1982), and plates were incubated at 32 °C for 2 days. Survival studies with mitomycin C and 4. nitroquinoline /.oxide Cells from log-phase cultures (4-10× l0 T cfu/ml) were washed by low-speed centrifugation with phosphate-buffered saline (PBS, 13.6 mM Na.,HPOJKH2PO 4, pH 7.0, 0.12 M NaCI), and suspended in the original volume of the same buffer. To portions of the suspension were added appropriate amounts of an aqueous solution of each chemical so as to give desired concentrations of the chemical, and the mixtures were incubated at 32 °C for 60 rain. Appropriate dilutions of the mixtures were made in PBS, and samples were spread on nutrient agar plates. Survival studies with 254-nm UV Log-phase cultures were diluted 2000-fold into PBS, and a 5-mi portion of the dilution was irradiated under a 5-W germicidal lamp at a dose rate of 0.12 j / m 2 / s . Samples were taken at inter. vals and spread on nutrient agar plates after dilution with PBS when appropriate.
45 Sun,ival studies with molecular photosensitization Cells were harvested from log-phase cultures by centrifugation and suspended in the original volume of PBS. The suspension was diluted 100fold into a 9: 1 (v/v) mixture of PBS and acetone, and left standing at room temperature for 5 min. It was then illuminated under a 312-rim light source (VL-30M, Vilber Lourmat, Marne la Vall6e, France) at a distance of 10 cm, and samples taken at intervals were spread on nutrient agar plates after dilution with PBS when needed. Acetone did not affect the colony-forming ability of the cells of each strain for at least 20 min at this concentration.
whereas strain DB7 is defective in UV endonuclease activity (Grossman, 1968; Okubo et al., 1971) as well as in the urrA homolog (Shiota and Nakayama, 1989). Hence, comparison of these strains should provide information about the effect of UV endonuclease on the UV survival of the cells as measured in the absence of interference by the Uvr homolog. For this comparison to be valid, however, it is required that both mutant strains be defective in the activity of the Uvr homolog to exactly the same degree. Unfortunately, such activity in wild-type M. luteus has not yet been detected in vitro. We therefore examined both strains for sensitivity to mitomycin C and 4-nitroquinoli,e 1-oxide. These chemicals are known to form adducts in DNA which are subject to repair by the E. coil Uvr system (Van Houten, 1990). Perhaps the M. luteus Uvr system can also repair such damage as judged by the sensitivity of U V ~ : I and DB7 to these chemicals (Shiota and Nakayama, 1988, 1989), whereas UV endonucle-
Results and discussion According to previous work, strain UVSN1 is mutant for the homolog of E. coil ul.'rB (Shiota and Nakayama, 1988) but normal with respect to UV endonuclease activity (Okubo et al., 1971),
i
ia
I Re---.~
^
b •
16 2 I
0
I
I
I
t
0.2
0.4
0.6
0.8
Mitomycin C ( gg ml-~ )
~
i
O
I
I
0.1
0°2
I
I
0.3
0.4
4-Nitroquinoline l-oxide (mM)
Fig. 1. Sensitivityof M. luteus strains to mitomycinC (a) or 4-nitroquinoline l-oxide (b). The results shown are those of typical experiments, o, DB7 (uL'rA. UV endonuclease-deficient);e, UV~NI (uurB, UV endonucleasenormal): zx, ATCC4698(wild type): A, uvr+ transformantof DB7 (UV endonuclease-deficient).
46
ase, which is highly specific for pyrimidine dimers, cannot. Consequently, it was anticipated that if the 2 strains are equivalent in the degree of Uvr deficiency, they should exhibit identical sensitivity to each of these chemicals irrespective of the presence or absence of UV endonuclease. This expectation was in fact realized as shown in Fig. 1. It should be noted that the equal sensitivity was seen with 2 different substances that are unrelated to each other in terms of chemical structure and the mechanism for enzymatic activation (Szybalski and Iyer, 1964, Tada, 1981). If one assumes that the defects of the Uvr homolog are different in degree between the 2 mutants, it must be compensated by a difference in some other factor, e.g., permeation or activation, for the identical sensitivity to result. It is highly unlikely that such compensation would occur with the 2 unrelated substances. We therefore conclude that the 2 strains have Uvr deficiencies
10-'
1(~2
0
60
120 180 Oose (sec)
240
300
Fig. 3. Sensitivity of M, luteus strains to molecular photosensitization, Survival rates are represented by means (symbols) and SDs (vertical bars) for 4 (UV'NI) or 5 (DBT) separate experiments. Abscissa. time of irradiation at 312 nm. o, DB?; e. UV~NI,
,I I
"~D 11~I |m
I f,4
l,
I
0
60
I
120 Dose (sec)
I
180
Fig. 2. Sensitivity of M, luteus strains to 254-nm UV light, Survival rates are represented by means (symbols) and SDs (vertical bars) of 4 separate experiments, Abscissa, time of irradiation, o, DB7; e. UV~NI,
functionally indistinguishable from each other. Perhaps both strains are 'null' mutants in view of the fact that DB7 has a nonsense mutation in the ut'rA homolog (Shiota and Nakayama, 1989). As shown in Fig. 2, DB7 was substantially more sensitive to 254-nm UV than UVSNI, with a dose reduction factor of around 2 at the survival level of 37%. In the light of the above-mentioned results with the chemicals, we considered it likely that this difference in UV survival reflected the presence or absence of UV endonuclease in these mutants. In order to strengthen this interpretation, we went on to look at the effect of molecular photosensitization with acetone as a sensitizer. Unlike 254-nm irradiation, which yields not only pyrimidine dimers but also a significant amount of 6-4 photoproducts, the acetone-mediated molecular photosensitization
47
has been shown to generate exclusively pyrimidine dimers (Ben-lshai, 1968). Consequently, if UV endonuclease is responsible for the differential killing of the 2 strains by 254-nm UV (Fig. 2), it was anticipated that the photosensitization ~'~uld reveal a greater strain difference in sensitivity. The results presented in Fig. 3 indicate that this was actually the case: DB7 was more sensitive to acetone plus 312-nm light than UV~N1, with an increased dose reduction facto( of about 3.4 at the 37% survival level. Incidentally, the wild-type strain and the UV endonuclease-deficient Uvr + transformant did not incur any loss of viability in the dose ranges shown in Figs. 2 and 3. From the above results, it seems highly likely that the difference in the sensitivity to 254-nm UV as well as to the acetone-mediated photosensitization between UV"NI and DB7 reflects the in vivo activity of UV endonuclease. Consistent with this idea is the observation by Tao et al. (1987) that their M. luteus mutants, which are similar to UV"NI, are invariably less UV-sensitire than, but as susceptible to adduct-forming chemicals as, DB7. As has previously been shown (Okubo etai., 1971; Shiota and Nakayama, 1989), a defect in UV endonuclease alone does not produce any appreciable effect on the sensitivity of the cell to 254-nm UV over a wide dose range. Unlike such M. luteus mutants, UV endonuclease (endonuclease V)-deficient (denV) mutants of phage T4 have long been known to be more UV-sensitire than the wild-type (Harm, 1963). Since T4 is insusceptible to host.cell reactivation (Ell(son et al., 1960), UV endonuclease should be the sole incision enzyme for this virus that is responsible for the excision repair of UV damage. Hence I"4 can now be regarded as equivalent to Uvr-deficient M. luteus mutants such as UV~N1, in which the incision reaction should also be dependent solely on UV endonuclease. We consider that the long-standing, apparent discrepancy between T4 and M. luteus with respect to the in vivo role for UV endonuclease has now been solved, with the unified view that UV endonuclease is functional in rive, with its contribution to UV survival being manifest only in the absence of another efficient incision system(s).
Acknowledgements We are grateful to Ms. C. Yoshida for her general supportive services in the laboratory. Technical help provided by Dr. K. Nakayama in an early phase of this work is acknowledged with appreciation.
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