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Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene
Mutation Research 459 Ž2000. 203–209 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres
Elevation of sister chromatid exchange in Saccharomyces cereÕisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom’s syndrome gene Fumitoshi Onoda a , Masayuki Seki a , Atsuko Miyajima b, Takemi Enomoto
a,)
a
b
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku UniÕersity, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan DiÕision of Pharmacology, Biological Safety Research Center, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan Received 9 September 1999; received in revised form 6 December 1999; accepted 10 December 1999
Abstract The SGS1 of Saccharomyces cereÕisiae is a homologue of the Bloom’s syndrome and Werner’s syndrome genes. The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation. In this study, we found that sister chromatid exchange was increased in sgs1 disruptants. We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity Ž sgs1-hd ., having equivalent missense mutations found in Bloom’s syndrome patients Ž sgs1-BS1, sgs1-BS2 .. None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants. On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: SGS1; Bloom’s syndrome; Werner’s syndrome; RecQ helicase; SCE
1. Introduction Bloom’s and Werner’s syndromes arise from mutations in the genes encoding a DNA helicase homologous to Escherichia coli RecQ which is a member of the RecF recombination pathway w1,2x. The representative clinical manifestations of Bloom’s syndrome ŽBS. are cancer predisposition, immunodeficiency, and male infertility w3,4x. The cells derived
) Corresponding author. Tel.: q81-22-217-6874; fax: q81-22217-6873; e-mail: [email protected]
from BS patients show increases in sensitivity to ethyl methanesulfonate, interchanges between homologous chromosomes, and sister chromatid exchange ŽSCE., which is an abnormality believed to be associated specifically with BS w3,5,6x. Werner’s syndrome ŽWS. patients prematurely develop a variety of major age-related diseases such as arteriosclerosis, malignant neoplasms, melituria, and cataract w7x. The cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening w8,9x. In addition to the two RecQ homologues, there exist three other RecQ homologues, RecQL1 ŽDNA
0921-8777r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 7 1 - 3
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helicase Q1rRecQL., RecQL4, and RecQL5 in human cells w10–12x. Recently, it has been reported that mutations in RECQL4 cause Rothmond–Thomson’s syndrome w13x. In contrast to higher eukaryotic cells, Saccharomyces cereÕisiae possesses only one RecQ homologue, Sgs1. A mutant allele of SGS1 was identified as a suppressor of the slow-growth phenotype of top3 mutants w14x. Two-hybrid experiments indicated that yeast Sgs1 interacts with DNA topoisomerase III w14x as well as DNA topoisomerase II w15x. The protein encoded by SGS1 has seven conserved helicase motifs and Sgs1 has been shown to actually possess DNA helicase activity w16,17x. Deletion mutants of SGS1 showed a reduction in the fidelity of chromosome segregation during mitosis and meiosis w15x, mitotic hyperrecombination phenotypes w18,19x, poor sporulation w15,18x and premature aging w20,21x. Thus sgs1 disruptants seem to become a good model for BS and WS. Although hyperrecombination phenotypes of sgs1 disruptants have been observed, including interchromosomal homologous recombination, intrachromosomal excision recombination, ectopic recombination, and illegitimate recombination w18,19x, it has not been examined whether SCE is elevated in sgs1 disruptants or not. To confirm the relevance of sgs1 disruptants as a model of BS, we examined in this study, the frequency of SCE in sgs1 disruptants and those introduced sgs1 genes containing equivalent missense mutations found in BS patients w1,22x, which we designated tentatively as sgs1-BS1 and sgs1-BS2. The BS1 allele ŽQ672R. is shown to code a protein defective in DNA helicase activity w23,24x. We also monitored methyl methanesulfonate ŽMMS. and hydroxyurea ŽHU . sensitivity, interchromosomal recombination and sporulation of sgs1 disruptants carrying sgs1-BS1 or sgs1-BS2 as well as those carrying the sgs1-hd, which was shown to code a Sgs1 lacking DNA helicase activity w16x. 2. Materials and methods 2.1. Yeast strains Yeast strains used in this study are as follows: MR202 Ž MAT ara ura3-52r ura3-52 leu2–3, 112
r leu2–3, 112 trp-289 r trp-289 his1-7 r his1-1 sgs1
F. Onoda et al.r Mutation Research 459 (2000) 203–209
205
Fig. 1. Sequence alignment of RecQ family helicases.
site-directed mutagenesis kit ŽStratagene.. The mutations were confirmed by DNA sequencing.
SC plates lacking His and Trp. After 3 days at 308C, the colonies were enumerated.
2.3. Analysis for MMS or HU sensitiÕity
2.6. Spoluration
Sensitivity to MMS or HU was assesed by diluting cells with distilled water and inoculating them on SC plates lacking tryptophan ŽTrp. or on SC plates lacking Trp, which contain various concentrations of MMS or HU. Cells were incubated for 3 days and survival was assessed by counting colonies.
Cells incubated on SC lacking Trp plate for 2 days were harvested, diluted with distilled water, and inoculated on SPO plates containing 1% potassium acetate, 0.1% yeast extract, 0.05% glucose. After an incubation for 5 days, sporulation was monitored with a phase-contrast microscope.
2.4. Detection of SCE
3. Results
The strain constructed for detecting unequal SCE has been described and diagrammed previously in detail w26x.
3.1. EleÕation of SCE frequency in sgs1 disruptants and inability of sgs1-BS1 and sgs1-BS2 to suppress the eleÕated SCE
2.5. Frequency of recombination between heteroalleles
We made sgs1 disruptants using the strain constructed for detecting unequal SCE w26x and plasmids
A strain, MR202, was constructed such that recombination between heteroalleles, his1-1r his1-7, in a diploid could be detected via a restoration of histidine ŽHis. prototrophy. Cells were inoculated on
Table 2 Suppression of increased SCE frequency in sgs1 disruptants by transformation with SGS1 genes
Table 1 Increase in the frequency of SCE in sgs1 disruptants Strain
Genotype
Recombination rate a
8202SCR FSCRs1
Wild type sgs1
31.24"6.868 236.7"74.10
a
The value indicates the number per 10 6 viable cells.
Plasmid
Mutation
Recombination rate a
pRS314 YCp1305 sgs1-hd sgs1-BS1 sgs1-BS2
vector only no mutation K706A Q683R C1047F
288.44"82.52 b 72.01"21.65 c 164.83"43.20 b,c 193.68"66.40 b,c 266.20"48.94 b,c
a
The value indicates the number per 10 6 viable cells. No significant difference Ž P ) 0.05.. c Significantly different from YCp1305 Ž P - 0.01.. b
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Fig. 2. MMS and HU sensitivity. The sgs1 disruptant ŽMR202. was transformed with pRS314 Žvector only, I. , pBLM1 Ž sgs1-BS1, e., pBLM2 Ž sgs1-BS2, ^., and pYCp1305 Ž SGS1, `.. Wild type ŽMR101. was transformed with pRS314 Žvector only, B., and pYCp1305 Ž SGS1, v .. Transformed cells were cultured in SC medium lacking Trp at 308C, diluted and inoculated on SC plates lacking Trp, which contained the indicated concentration of MMS or HU.
containing the sgs1 gene carrying equivalent missense mutations of those found in BS patients, sgs1BS1 and sgs1-BS2. The BS1 allele has a missense mutation of the glutamine residue ŽQ672R., which is conserved within DNArRNA helicase families including RecQ helicase family w1,27x ŽFig. 1.. BS2 has a missense mutation of the cysteine residue ŽC1047F. conserved within RecQ helicase family w22x. We also constructed the sgs1-hd, which has been confirmed to encode a Sgs1 lacking DNA helicase activity w16x. As shown in Table 1, the frequency of spontaneous SCE is elevated by 4-fold in sgs1 disruptants as compared to that of wild-type cells. The elevated SCE in sgs1 disruptants was suppressed by wild-type SGS1 but not by sgs1-BS1, sgs1-BS2, or sgs1-hd ŽTable 2.. Because the data in the table were subjected to statistical analysis for differences, the significance of the differences in SCE rates between pRS314 and mutated SGS1 Ž sgs1-hd, sgs1-BS1, sgs1-BS2 . transformed cells could not be proven on a 5% level.
HU of sgs1 disruptants. When sgs1 disruptants were transformed with a plasmid containing wild-type SGS1, they became resistant to MMS and HU at the levels of wild-type cells ŽFig. 2.. As expected, sgs1hd behaved like as an empty vector. Neither sgs1BS1 nor sgs1-BS2 could suppress MMS or HU sensitivity.
3.2. sgs1-BS1 and sgs1-BS2 alleles could not suppress the sensitiÕities to MMS and HU and the increased frequency of interchromosomal recombination of sgs1 disruptants
Fig. 3. Heteroallelic recombination. The sgs1 disruptant ŽMR202. was transformed with pRS314 Žvector only., pBLM1 Ž sgs1-BS1., pBLM2 Ž sgs1-BS2 ., and pYCp1305 Ž SGS1.. Transformed cells were cultured in SC medium lacking Trp at 308C and diluted. Aliquots were inoculated on YPAD plates and 10 6 cells were inoculated onto SC plates lacking Trp and His, and incubated at 308C for 4 days. The value indicates the number of recombinants per 10 6 viable cells.
We next examined the ability of sgs1-BS1 and sgs1-BS2 to suppress the sensitivities to MMS and
F. Onoda et al.r Mutation Research 459 (2000) 203–209 Table 3 Restoration of spore forming ability in sgs1 disruptants by mutated sgs1 genes Plasmid
pRS314
YCp1305
sgs1-BS1
sgs1-BS2
Sporulation Ž%.
7.14
68.13
40.78
39.76
Since the increase in the frequency of recombination between heteroallelles is demonstrated, not only in BS cells w3x, but also in sgs1 disruptants w8x, we examined the effect of sgs1-BS1 and sgs1-BS2 on the frequency of recombination between heteroallelles. As shown in Fig. 3, sgs1-BS1 and sgs1-BS2 as well as sgs1-hd could not suppress spontaneously elevated interchromosomal recombinations in sgs1 disruptants. 3.3. sgs1-BS1 and sgs1-BS2 alleles complement poor sporulation phenotype of sgs1 disruptants It has been reported that the sgs1-hd allele behaves just like as wild-type one: it decreases the growth rate of top3-sgs1 mutants and improves the poor growth of top1-sgs1 mutants w16x. In addition, we found that poor sporulation phenotype of sgs1 disruptants was complemented by the sgs1-hd allele Žunpublished data.. Thus, we examined whether the poor sporulation phenotype is complemented by sgs1-BS1 and sgs1-BS2. As shown in Table 3, sgs1BS1 and sgs1-BS2 as well as sgs1-hd suppressed the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wildtype SGS1.
4. Discussion In this study, we have shown that SCE frequency is elevated in sgs1 disruptants as in BS cells and that sgs1 having the same missense mutations found in BS cannot suppress the increase in the frequency of SCE, indicating the relevance of sgs1 disruptants as a model of BS. The results obtained by using sgs1-hd indicated that DNA helicase activity of Sgs1 is required for suppression of SCE and interchromosomal recombinations as well as for complementation of MMS and
207
HU sensitivity. The inability of sgs1-BS1 and sgs1BS2 to suppress SCE and interchromosomal recombinations and to complement MMS and HU sensitivity indicates that proteins coded by these alleles have a defect in DNA helicase activity. In fact, the missense mutation of BS1 has been shown to abolish DNA helicase activity w23,24x. sgs1-BS1 and sgs1-BS2 as well as sgs1-hd complemented the poor sporulation phenotype of sgs1 disruptants, indicating that the products coded by sgs1-BS1 and sgs1-BS2 could take a configuration to support sporulation during meiosis. It has been reported that the sgs1-hd allele behaves just like the wild-type, decreasing the growth rate of top3-sgs1 mutants w16x. This result raises the possibility that the function of Sgs1 required for sporulation is related to the function of DNA topoisomerase III. Recently, it was shown that yeast DNA topoisomerase III is required for the meiotic recombination w28x. Thus it seems likely that one of the functions of Sgs1 is to recruit topoisomerase III to meiotic chromosomes. We observed that mouse Blm, Top3a, and Top3b mRNAs are highly expressed in the testis w29–31x. The recent immunocytological study on mouse spermatocytes shows that the Blm is first evident as discrete foci along the synaptonemal complexes of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. Blm foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema w32x. It was suggested that Blm is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase and involved in resolution of interlocks, which requires the function of topoisomerase, in preparation for homologous chromosome disjunction during anaphase I w32x. If the molecular mechanisms underlying poor sporulation phenotype of yeast sgs1 disruptants and hypogonadism of BS patients are the same, it can be speculated that the BS patients having BS1 or BS2 mutation are fertile. However, almost all BS patients show hypogonadism because most of mutations identified in these patients cause premature termination of translation w1x, which results in the impaired nuclear import of proteins, due to the existence of the nuclear localization signal in the carboxy terminus of BLM w33x.
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5. Conclusion We have shown that sgs1 disruptants provide a good model system for analyzing the functions of BLM. From the results obtained by using this system, we can speculate that the increase in SCE frequency and the hypogonadism of BS patients are due to different defects in BLM functions.
w11x
w12x
Acknowledgements We greatly acknowledge L.H. Hartwell for the supply of 8202SCR. This research was supported by Grants-in Aid for Scientific Research ŽB., for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and the Mitsubishi Foundation.
w13x
w14x
w15x
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w30x T. Seki, M. Seki, T. Katada, T. Enomoto, Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis, Biochim. Biophys. Acta 1396 Ž1998. 127–131. w31x T. Seki, M. Seki, R. Onodera, T. Katada, T. Enomoto, Cloning of cDNA encoding a novel mouse DNA topoisomerase III ŽTopo IIIbeta. possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis, J. Biol. Chem. 273 Ž1998. 28553–28556. w32x D. Walpita, A.W. Plug, N.F. Neff, J. German, T. Ashley, Bloom’s syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes, Proc. Natl. Acad. Sci. U.S.A. 96 Ž1999. 5622–5627. w33x H. Kaneko, K.O. Orii, E. Matsui, N. Shimozawa, T. Fukao, T. Matsumoto, A. Shimamoto, Y. Furuichi, S. Hayakawa, K. Kasahara, N. Kondo, BLM Žthe causative gene of Bloom syndrome. protein translocation into the nucleus by a nuclear localization signal, Biochem. Biophys. Res. Commun. 240 Ž1997. 348–353.
Elevation of sister chromatid exchange in Saccharomyces cereÕisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom’s syndrome gene Fumitoshi Onoda a , Masayuki Seki a , Atsuko Miyajima b, Takemi Enomoto
a,)
a
b
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku UniÕersity, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan DiÕision of Pharmacology, Biological Safety Research Center, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan Received 9 September 1999; received in revised form 6 December 1999; accepted 10 December 1999
Abstract The SGS1 of Saccharomyces cereÕisiae is a homologue of the Bloom’s syndrome and Werner’s syndrome genes. The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation. In this study, we found that sister chromatid exchange was increased in sgs1 disruptants. We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity Ž sgs1-hd ., having equivalent missense mutations found in Bloom’s syndrome patients Ž sgs1-BS1, sgs1-BS2 .. None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants. On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: SGS1; Bloom’s syndrome; Werner’s syndrome; RecQ helicase; SCE
1. Introduction Bloom’s and Werner’s syndromes arise from mutations in the genes encoding a DNA helicase homologous to Escherichia coli RecQ which is a member of the RecF recombination pathway w1,2x. The representative clinical manifestations of Bloom’s syndrome ŽBS. are cancer predisposition, immunodeficiency, and male infertility w3,4x. The cells derived
) Corresponding author. Tel.: q81-22-217-6874; fax: q81-22217-6873; e-mail: [email protected]
from BS patients show increases in sensitivity to ethyl methanesulfonate, interchanges between homologous chromosomes, and sister chromatid exchange ŽSCE., which is an abnormality believed to be associated specifically with BS w3,5,6x. Werner’s syndrome ŽWS. patients prematurely develop a variety of major age-related diseases such as arteriosclerosis, malignant neoplasms, melituria, and cataract w7x. The cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening w8,9x. In addition to the two RecQ homologues, there exist three other RecQ homologues, RecQL1 ŽDNA
0921-8777r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 7 1 - 3
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helicase Q1rRecQL., RecQL4, and RecQL5 in human cells w10–12x. Recently, it has been reported that mutations in RECQL4 cause Rothmond–Thomson’s syndrome w13x. In contrast to higher eukaryotic cells, Saccharomyces cereÕisiae possesses only one RecQ homologue, Sgs1. A mutant allele of SGS1 was identified as a suppressor of the slow-growth phenotype of top3 mutants w14x. Two-hybrid experiments indicated that yeast Sgs1 interacts with DNA topoisomerase III w14x as well as DNA topoisomerase II w15x. The protein encoded by SGS1 has seven conserved helicase motifs and Sgs1 has been shown to actually possess DNA helicase activity w16,17x. Deletion mutants of SGS1 showed a reduction in the fidelity of chromosome segregation during mitosis and meiosis w15x, mitotic hyperrecombination phenotypes w18,19x, poor sporulation w15,18x and premature aging w20,21x. Thus sgs1 disruptants seem to become a good model for BS and WS. Although hyperrecombination phenotypes of sgs1 disruptants have been observed, including interchromosomal homologous recombination, intrachromosomal excision recombination, ectopic recombination, and illegitimate recombination w18,19x, it has not been examined whether SCE is elevated in sgs1 disruptants or not. To confirm the relevance of sgs1 disruptants as a model of BS, we examined in this study, the frequency of SCE in sgs1 disruptants and those introduced sgs1 genes containing equivalent missense mutations found in BS patients w1,22x, which we designated tentatively as sgs1-BS1 and sgs1-BS2. The BS1 allele ŽQ672R. is shown to code a protein defective in DNA helicase activity w23,24x. We also monitored methyl methanesulfonate ŽMMS. and hydroxyurea ŽHU . sensitivity, interchromosomal recombination and sporulation of sgs1 disruptants carrying sgs1-BS1 or sgs1-BS2 as well as those carrying the sgs1-hd, which was shown to code a Sgs1 lacking DNA helicase activity w16x. 2. Materials and methods 2.1. Yeast strains Yeast strains used in this study are as follows: MR202 Ž MAT ara ura3-52r ura3-52 leu2–3, 112
r leu2–3, 112 trp-289 r trp-289 his1-7 r his1-1 sgs1
F. Onoda et al.r Mutation Research 459 (2000) 203–209
205
Fig. 1. Sequence alignment of RecQ family helicases.
site-directed mutagenesis kit ŽStratagene.. The mutations were confirmed by DNA sequencing.
SC plates lacking His and Trp. After 3 days at 308C, the colonies were enumerated.
2.3. Analysis for MMS or HU sensitiÕity
2.6. Spoluration
Sensitivity to MMS or HU was assesed by diluting cells with distilled water and inoculating them on SC plates lacking tryptophan ŽTrp. or on SC plates lacking Trp, which contain various concentrations of MMS or HU. Cells were incubated for 3 days and survival was assessed by counting colonies.
Cells incubated on SC lacking Trp plate for 2 days were harvested, diluted with distilled water, and inoculated on SPO plates containing 1% potassium acetate, 0.1% yeast extract, 0.05% glucose. After an incubation for 5 days, sporulation was monitored with a phase-contrast microscope.
2.4. Detection of SCE
3. Results
The strain constructed for detecting unequal SCE has been described and diagrammed previously in detail w26x.
3.1. EleÕation of SCE frequency in sgs1 disruptants and inability of sgs1-BS1 and sgs1-BS2 to suppress the eleÕated SCE
2.5. Frequency of recombination between heteroalleles
We made sgs1 disruptants using the strain constructed for detecting unequal SCE w26x and plasmids
A strain, MR202, was constructed such that recombination between heteroalleles, his1-1r his1-7, in a diploid could be detected via a restoration of histidine ŽHis. prototrophy. Cells were inoculated on
Table 2 Suppression of increased SCE frequency in sgs1 disruptants by transformation with SGS1 genes
Table 1 Increase in the frequency of SCE in sgs1 disruptants Strain
Genotype
Recombination rate a
8202SCR FSCRs1
Wild type sgs1
31.24"6.868 236.7"74.10
a
The value indicates the number per 10 6 viable cells.
Plasmid
Mutation
Recombination rate a
pRS314 YCp1305 sgs1-hd sgs1-BS1 sgs1-BS2
vector only no mutation K706A Q683R C1047F
288.44"82.52 b 72.01"21.65 c 164.83"43.20 b,c 193.68"66.40 b,c 266.20"48.94 b,c
a
The value indicates the number per 10 6 viable cells. No significant difference Ž P ) 0.05.. c Significantly different from YCp1305 Ž P - 0.01.. b
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Fig. 2. MMS and HU sensitivity. The sgs1 disruptant ŽMR202. was transformed with pRS314 Žvector only, I. , pBLM1 Ž sgs1-BS1, e., pBLM2 Ž sgs1-BS2, ^., and pYCp1305 Ž SGS1, `.. Wild type ŽMR101. was transformed with pRS314 Žvector only, B., and pYCp1305 Ž SGS1, v .. Transformed cells were cultured in SC medium lacking Trp at 308C, diluted and inoculated on SC plates lacking Trp, which contained the indicated concentration of MMS or HU.
containing the sgs1 gene carrying equivalent missense mutations of those found in BS patients, sgs1BS1 and sgs1-BS2. The BS1 allele has a missense mutation of the glutamine residue ŽQ672R., which is conserved within DNArRNA helicase families including RecQ helicase family w1,27x ŽFig. 1.. BS2 has a missense mutation of the cysteine residue ŽC1047F. conserved within RecQ helicase family w22x. We also constructed the sgs1-hd, which has been confirmed to encode a Sgs1 lacking DNA helicase activity w16x. As shown in Table 1, the frequency of spontaneous SCE is elevated by 4-fold in sgs1 disruptants as compared to that of wild-type cells. The elevated SCE in sgs1 disruptants was suppressed by wild-type SGS1 but not by sgs1-BS1, sgs1-BS2, or sgs1-hd ŽTable 2.. Because the data in the table were subjected to statistical analysis for differences, the significance of the differences in SCE rates between pRS314 and mutated SGS1 Ž sgs1-hd, sgs1-BS1, sgs1-BS2 . transformed cells could not be proven on a 5% level.
HU of sgs1 disruptants. When sgs1 disruptants were transformed with a plasmid containing wild-type SGS1, they became resistant to MMS and HU at the levels of wild-type cells ŽFig. 2.. As expected, sgs1hd behaved like as an empty vector. Neither sgs1BS1 nor sgs1-BS2 could suppress MMS or HU sensitivity.
3.2. sgs1-BS1 and sgs1-BS2 alleles could not suppress the sensitiÕities to MMS and HU and the increased frequency of interchromosomal recombination of sgs1 disruptants
Fig. 3. Heteroallelic recombination. The sgs1 disruptant ŽMR202. was transformed with pRS314 Žvector only., pBLM1 Ž sgs1-BS1., pBLM2 Ž sgs1-BS2 ., and pYCp1305 Ž SGS1.. Transformed cells were cultured in SC medium lacking Trp at 308C and diluted. Aliquots were inoculated on YPAD plates and 10 6 cells were inoculated onto SC plates lacking Trp and His, and incubated at 308C for 4 days. The value indicates the number of recombinants per 10 6 viable cells.
We next examined the ability of sgs1-BS1 and sgs1-BS2 to suppress the sensitivities to MMS and
F. Onoda et al.r Mutation Research 459 (2000) 203–209 Table 3 Restoration of spore forming ability in sgs1 disruptants by mutated sgs1 genes Plasmid
pRS314
YCp1305
sgs1-BS1
sgs1-BS2
Sporulation Ž%.
7.14
68.13
40.78
39.76
Since the increase in the frequency of recombination between heteroallelles is demonstrated, not only in BS cells w3x, but also in sgs1 disruptants w8x, we examined the effect of sgs1-BS1 and sgs1-BS2 on the frequency of recombination between heteroallelles. As shown in Fig. 3, sgs1-BS1 and sgs1-BS2 as well as sgs1-hd could not suppress spontaneously elevated interchromosomal recombinations in sgs1 disruptants. 3.3. sgs1-BS1 and sgs1-BS2 alleles complement poor sporulation phenotype of sgs1 disruptants It has been reported that the sgs1-hd allele behaves just like as wild-type one: it decreases the growth rate of top3-sgs1 mutants and improves the poor growth of top1-sgs1 mutants w16x. In addition, we found that poor sporulation phenotype of sgs1 disruptants was complemented by the sgs1-hd allele Žunpublished data.. Thus, we examined whether the poor sporulation phenotype is complemented by sgs1-BS1 and sgs1-BS2. As shown in Table 3, sgs1BS1 and sgs1-BS2 as well as sgs1-hd suppressed the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wildtype SGS1.
4. Discussion In this study, we have shown that SCE frequency is elevated in sgs1 disruptants as in BS cells and that sgs1 having the same missense mutations found in BS cannot suppress the increase in the frequency of SCE, indicating the relevance of sgs1 disruptants as a model of BS. The results obtained by using sgs1-hd indicated that DNA helicase activity of Sgs1 is required for suppression of SCE and interchromosomal recombinations as well as for complementation of MMS and
207
HU sensitivity. The inability of sgs1-BS1 and sgs1BS2 to suppress SCE and interchromosomal recombinations and to complement MMS and HU sensitivity indicates that proteins coded by these alleles have a defect in DNA helicase activity. In fact, the missense mutation of BS1 has been shown to abolish DNA helicase activity w23,24x. sgs1-BS1 and sgs1-BS2 as well as sgs1-hd complemented the poor sporulation phenotype of sgs1 disruptants, indicating that the products coded by sgs1-BS1 and sgs1-BS2 could take a configuration to support sporulation during meiosis. It has been reported that the sgs1-hd allele behaves just like the wild-type, decreasing the growth rate of top3-sgs1 mutants w16x. This result raises the possibility that the function of Sgs1 required for sporulation is related to the function of DNA topoisomerase III. Recently, it was shown that yeast DNA topoisomerase III is required for the meiotic recombination w28x. Thus it seems likely that one of the functions of Sgs1 is to recruit topoisomerase III to meiotic chromosomes. We observed that mouse Blm, Top3a, and Top3b mRNAs are highly expressed in the testis w29–31x. The recent immunocytological study on mouse spermatocytes shows that the Blm is first evident as discrete foci along the synaptonemal complexes of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. Blm foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema w32x. It was suggested that Blm is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase and involved in resolution of interlocks, which requires the function of topoisomerase, in preparation for homologous chromosome disjunction during anaphase I w32x. If the molecular mechanisms underlying poor sporulation phenotype of yeast sgs1 disruptants and hypogonadism of BS patients are the same, it can be speculated that the BS patients having BS1 or BS2 mutation are fertile. However, almost all BS patients show hypogonadism because most of mutations identified in these patients cause premature termination of translation w1x, which results in the impaired nuclear import of proteins, due to the existence of the nuclear localization signal in the carboxy terminus of BLM w33x.
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5. Conclusion We have shown that sgs1 disruptants provide a good model system for analyzing the functions of BLM. From the results obtained by using this system, we can speculate that the increase in SCE frequency and the hypogonadism of BS patients are due to different defects in BLM functions.
w11x
w12x
Acknowledgements We greatly acknowledge L.H. Hartwell for the supply of 8202SCR. This research was supported by Grants-in Aid for Scientific Research ŽB., for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and the Mitsubishi Foundation.
w13x
w14x
w15x
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