- Email: [email protected]
Targeted Deletion of a GA Tract by S1 Nuclease
445
NOTES & TIPS
sistent with Resource Q anion-exchange HPLC and light scattering analyses (data not shown). Also from the table, either lowering the pH to 6.5 or increasing it to pH 8 and 8.5 resulted in slightly more free DNA detected, suggesting that particular pH shifts could effect some subtle structural changes that would allow some dye to be internalized into the virus. Furthermore, although the amount of free DNA detected at 387C is similar to that at 227C, the amount of free DNA for the sample subjected to 807C was high and appears to be more than the total expected DNA in the virions. This could be due to fluorescence enhancement of DNA–dye complexes trapped in hydrophobic pockets or concentration effects as a result of solution evaporation. Discussion. In this study, we have developed an automated microfluorometric assay using the H33258 dye for DNA quantitation. The assay was ideal for the adenovirus model, where detection and quantitation of free DNA from unpackaged viral DNA, host cell DNA, or DNA present in virus assembly intermediates was possible without picking up the DNA packaged in the mature rigid virions. The assay takes advantage of the small 16-mL flow cell of the Waters 474 HPLC fluorescence detector, the autoinjection system, and the high sensitivity and specificity of H33258 dye for DNA detection. The result was a simple, self-contained, 2-min, automated procedure that requires low volumes of sample and that has a sensitivity of detection of 1 ng DNA, similar to that for the capillary cuvette assay and the microwell assay. The assay strategy is certainly applicable to absorbance-based assays and all other conceivable analyses based on some physicochemical property of an analyte. The level of free DNA was also studied in adenoviral samples under critical environmental factors such as ionic strength, pH, and temperature. Care should be taken in quantitating the amount of DNA of a particular sample, since the enhancement of H33258 fluorescence has been found to be proportional to the AT content (9). The background free DNA (5%) for the starting material could be actual free DNA, DNA in association with empty capsids (10), DNA inside any of the virus assembly intermediates, or DNA inside a young virion; the assembly intermediates and the young virion have structures that are permeable to nucleases and low MW compounds such as H33258 (11). Further studies have to be done to confirm the actual contributing factor(s). The components of the viral capsid are held together by ionic and hydrophobic interactions and further stabilized by structural proteins; disruption of these interactions by increased ionic strength, gross changes in pH, and high temperatures result to the detection of more free DNA, which certainly impact on the stability and consequently on the efficacy of the
gene vector. Finally, the assay can be a standard for monitoring the levels of free DNA in recombinant proteins and viruses earmarked for commercial production of human therapeutics. Acknowledgments. The authors thank Mr. Andres Frei, Dr. Vijay Singh, Dr. Ronald Bordens, and Dr. Angel Cruz for encouragement and valuable suggestions and comments. This research was supported by funds granted by the Schering-Plough Research Institute.
REFERENCES 1. Henry, L. J., Xia, D., Wilke, M. E., Deisenhofer, J., and Gerard, R. D. (1994) J. Virol. 68, 5239–5246. 2. Philipson, L., and Lindberg, U. (1974) in Comprehensive Virology (Frankel-Conrat, H., and Wagner, R. R., Eds.), Vol. 3, pp. 143–227, Plenum, New York. 3. Cesarone, C. F., Bolognesi, C., and Santi, L. (1979) Anal. Biochem. 100, 188–197. 4. Labarca, C., and Paigen, K. (1980) Anal. Biochem. 102, 344– 352. 5. Huyghe, B. G., Liu, X., Sutjipto, S., Sugarman, B. J., Horn, M. T., Shepard, H. M., Scandella, C. J., and Shabram, P. (1995) Hum. Gene Ther. 6, 1403–1416. 6. Maizel, J. V., Jr., White, D. O., and Scharff, M. D. (1968) Virology 36, 115–125. 7. Green, M., and Pinˇa, M. (1963) Virology 20, 199–207. 8. Gallagher, S., and Rymaszewski, Z. (1994) Methods Toxicol. 1B, 164–177. 9. Loontiens, F. G., Regenfuss, P., Zechel, A., Dumortier, L., and Clagg, R. M. (1990) Biochemistry 29, 9029–9039. 10. Tibbetts, C., and Giam, C.-Z. (1979) J. Virol. 32(3), 995–1005. 11. Horwitz, M. S. (1990) in Virology (Fields, B. N., Knipe, D. M., et al., Eds.), 2nd ed., pp. 1679–1721, Raven Press, New York.
Targeted Deletion of a GA Tract by S1 Nuclease Benoit Barbeau and Eric Rassart1 De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, Montre´al, Que´bec, Canada H3C 3P8 Received October 18, 1996
Purine/pyrimidine sequences are abundant in eukaryotic genomic DNA. Certain of these DNA regions contain purine/pyrimidine mirror repeats and can show sensitity to S1 nuclease. Such regions are strongly indicative for the presence of a DNA structure known as H-DNA. This latter structure is characterized by the formation of a triple-stranded helix and a S1 nuclease1 To whom correspondence should be addressed at De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, C.P.8888 Succ. Centre-ville, Montre´al, PQ, Canada H3C 3P8. Fax: (514) 9874647.
ANALYTICAL BIOCHEMISTRY 247, 445–447 (1997) ARTICLE NO. AB972085
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
04-15-97 13:03:18
abnt
446
NOTES & TIPS
FIG. 1. Plasmid pSH2-7CAT compared to the deleted mutants. (A) 1500 bp of 5* flanking sequence was positioned upstream of the CAT gene (indicated by an arrow) (7) (GenBank Accession No. L47616). The 133-nt purine-rich segment is highlighted by a dark rectangle in the flanking region and the 24 GA repeats are presented below. The oligonucleotide B14 (small arrow) is positioned at the 5* end of the CAT gene in an inverted orientation. Relative distance of the presented map is indicated for the size of 200 bp. Both restriction sites BamHI and SacI are shown above the map. (B) S1 nuclease-treated clones 10S1-5, 50S1-1, and 10S1-4 were selected and sequenced using the oligonucleotide B14. Part of the purine-rich region is presented and includes the GA repeat. Deleted nucleotides for each mutant plasmids are indicated by a dash. Positioning of the deleted region is based on sequence comparison with the parental pSH2-7CAT plasmid.
sensitive single-stranded DNA segment. The formation of H-DNA is dependent on parameters such as pH, DNA supercoiling, and ion concentration (1–3). Although present in many different genomic regions, HDNA has been observed in several promoters, especially those categorized as TATA-less promoters (4, 5). Furthermore, it has been reported that H-DNA could block transcription (6). In order to assess the potential role of GA repeats (and thus potential H-DNA structure) in gene regulation, we have developed a protocol of targeted deletion of GA repeat structure using S1 nuclease, thus taking advantage of the presence of the potential H-DNA single-stranded region located in this segment. The plasmid used in this set of experiments is pSH2-7CAT and contains 1500 bp of the human 5* flanking region of the Fli-1 locus upstream of the CAT gene. A conserved GA repeat region made of 24 repeats lies near the 3* end of the 5* flanking region. This GA motif is present in a very purine-rich region (93%) of 133 bp (Fig. 1A). We have previously shown that the corresponding region of the mouse Fli-1 promoter region is S1 nuclease sensitive and thus suggestively adopts an H-DNA conformation (7). Circular pSH2-7 CAT plasmid (1 mg) was first treated with S1 nuclease following a modified version of the protocol of Johnson et al. (8). S1 nuclease treatment was performed for 25 min at 427C with increasing amounts of S1 nuclease (10, 20, 50, 100, and 200 units) (Pharmacia, Montreal, Canada) in 30 ml of 30 mM
NaOAc, pH 4.5, 300 mM NaCl, 0.2 mM EDTA, and 3 mM ZnCl2 . DNA samples were then extracted once with phenol–chloroform and separated with a 1% agarose gel. DNA fragments corresponding to linearized plasmids were extracted from the gel by filtering of the agarose plug through a Whatman paper to be finally ethanol precipitated. Next, the purified DNA was treated with 6 units of Klenow DNA polymerase (Pharmacia) in 20 ml 11 One-Phor-All buffer containing 250 mM dNTP for 30 min at 377C. After heat inactivation of the enzyme, 2 ml of the treated DNA was used for self-ligation in a final volume of 20 ml ligase buffer 11 (BRL) with 1 U T4 DNA ligase (Gibco BRL, Burlington, Canada) for 16 h at room temperature. Five microliters of the ligated product was used for DH5-a transformation. Bacterial clones containing high-molecularweight plasmid DNA were then selected. Sequence analysis was performed with the T7 Pharmacia sequencing kit (Pharmacia) and the oligonucleotide B14 (5*-GTGGTATATCCAGTGATTTT-3*) localized 40 bp from the 5* end of the CAT gene in the antisense strand (Fig. 1A). Of 20 bacterial clones, 3 were further selected for their plasmid DNA which showed targeted deletion of 12 to 13 GA repeats (clones 10S1-4 and 10S1-5 generated after a 10 U S1 treatment and clone 50S11 generated after a 50 U S1 treatment; Fig. 1B). Indeed, sequence analysis indicated that clones 10S15 and 10S1-4 had a deletion of the 5* half of the GA motif and included 7 and 28 bp of 5* flanking
AID
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/
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NOTES & TIPS
447
FIG. 2. Comparison of S1 nuclease sensitivity between pSH2-7CAT and deleted mutant plasmids. The parental plasmid pSH2-7CAT (A) and the deleted clones 10S1-4 (B) and 50S1-1 (C) were treated with different amounts of S1 nuclease (lane 2, 10 U; lane 3, 20 U; lane 4, 50 U; lane 5, 100 U; lane 6, 200 U) and separated with a 1% agarose gel. Comparison of S1 nuclease treatment can be made with the untreated sample (ND) for each plasmids. A linearized BamHI-digested pSH2-7CAT plasmid is also included in A. (R, relaxed; L, linear; S, supercoiled).
sequence, respectively (Fig. 1B). On the other hand, clone 50S1-1 had a deletion of the 3 * half of the GA motif and only included 1 bp downstream of the GA repeat (Fig. 1B). The two different types of GA repeat deletions (5* end and 3 * end) could be reminiscent of the presence of two different H-DNA isomers for the plasmid pSH2-7CAT. The specificity of the deletion was further confirmed by comparison of the restriction maps of the deleted and the wild-type plasmids, which showed no difference outside of the targeted region; in addition, no other apparent deletion in the surrounding sequences could be observed (data not shown). However, it should be emphasized that other types of non-B-DNA structures have not been investigated in this study and thus we cannot rule out that such other structures might also be sensitive to the described deletion protocol. To evaluate whether the pSH2-7CAT-derived mutants were less susceptible to S1 treatment, clones 10S1-4, 50S1-1, and the parental plasmid pSH27CAT were again treated with increasing amounts of S1 nuclease (Fig. 2). When comparisons of the intensity ratio between linear and relaxed forms were performed, plasmid pSH2-7CAT treated with 10 U S1 nuclease could be matched with clones 10S1-4 and 50S1-1 treated with 100 U and 50 U S1 nuclease, respectively (Fig. 2). These results thus demonstrate that the removal of GA repeat seems to have greatly reduced or even possibly abolished S1 sensitivity of this region in the pSH2-7 CAT plasmid and most likely renders the region less prone to form an HDNA structure. Although the actual mode by which complete S1 nuclease DNA cleavage is achieved has not been clarified in this study, several possibilities can be suggested including the targeting of the unpaired single-stranded region of H-DNA structure as well as the previously proposed S1 nuclease sensitivity of the Hoogsteen base
AID
AB $NAT
/
6m2f$$$562
04-15-97 13:03:18
pair in the triple-stranded helix (9). Several methods of deletion have been described and include exonuclease treatment (for example, BAL31, DNaseI, or exoIII) (10) and PCR-based deletion protocols (11). Unlike these other techniques, our method seemingly targets region of potential H-DNA structures and could thus help deciphering the role played by these latter in different promoters. Acknowledgments. We gratefully acknowledge Richard Bergeron for excellent technical assistance. B.B. was supported by a Quebec FCAR scholarship. This work was supported by the Medical Research Council of Canada (Grant MT7754).
REFERENCES 1. Hanvey, J. C., Shimizu, M., and Wells, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 6292–6295. 2. Kohwi, Y., and Kohwi-Shigematsu, T. (1993) J. Mol. Biol. 231, 1090–1101. 3. Mirkin, S. M., and Frank-Kamenetskii, M. D. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 541–576. 4. Firulli, A. B., Maibenco, D. C., and Kinniburgh, A. J. (1992) Biochem. Biophys. Res. Commun. 185, 264–270. 5. Pestov, D. G., Dayn, A., Siyanova, E. Y., George, D. L., and Mirkin, S. M. (1991) Nucleic Acids Res. 19, 6527–5632. 6. Grabczyk, E., and Fishman, M. C. (1995) J. Biol. Chem. 270, 1791–1797. 7. Barbeau, B., Bergeron, D., Beaulieu, M., Nadjem, Z., and Rassart, E. (1996) Biochim. Biophys. Acta 1307, 220–232. 8. Johnson, A. C., Jinno, Y., and Merlino, G. T. (1988) Mol. Cell Biol. 8, 4174–4184. 9. Pulleyblank, D. E., Haniford, D. B., and Morgan, R. (1985) Cell 42, 271–280. 10. Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual. 2th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 11. Vallette, F., Mege, E., Reiss, A., and Adesnik, M. (1989) Nucleic Acids Res. 17, 723–733.
abnt
NOTES & TIPS
sistent with Resource Q anion-exchange HPLC and light scattering analyses (data not shown). Also from the table, either lowering the pH to 6.5 or increasing it to pH 8 and 8.5 resulted in slightly more free DNA detected, suggesting that particular pH shifts could effect some subtle structural changes that would allow some dye to be internalized into the virus. Furthermore, although the amount of free DNA detected at 387C is similar to that at 227C, the amount of free DNA for the sample subjected to 807C was high and appears to be more than the total expected DNA in the virions. This could be due to fluorescence enhancement of DNA–dye complexes trapped in hydrophobic pockets or concentration effects as a result of solution evaporation. Discussion. In this study, we have developed an automated microfluorometric assay using the H33258 dye for DNA quantitation. The assay was ideal for the adenovirus model, where detection and quantitation of free DNA from unpackaged viral DNA, host cell DNA, or DNA present in virus assembly intermediates was possible without picking up the DNA packaged in the mature rigid virions. The assay takes advantage of the small 16-mL flow cell of the Waters 474 HPLC fluorescence detector, the autoinjection system, and the high sensitivity and specificity of H33258 dye for DNA detection. The result was a simple, self-contained, 2-min, automated procedure that requires low volumes of sample and that has a sensitivity of detection of 1 ng DNA, similar to that for the capillary cuvette assay and the microwell assay. The assay strategy is certainly applicable to absorbance-based assays and all other conceivable analyses based on some physicochemical property of an analyte. The level of free DNA was also studied in adenoviral samples under critical environmental factors such as ionic strength, pH, and temperature. Care should be taken in quantitating the amount of DNA of a particular sample, since the enhancement of H33258 fluorescence has been found to be proportional to the AT content (9). The background free DNA (5%) for the starting material could be actual free DNA, DNA in association with empty capsids (10), DNA inside any of the virus assembly intermediates, or DNA inside a young virion; the assembly intermediates and the young virion have structures that are permeable to nucleases and low MW compounds such as H33258 (11). Further studies have to be done to confirm the actual contributing factor(s). The components of the viral capsid are held together by ionic and hydrophobic interactions and further stabilized by structural proteins; disruption of these interactions by increased ionic strength, gross changes in pH, and high temperatures result to the detection of more free DNA, which certainly impact on the stability and consequently on the efficacy of the
gene vector. Finally, the assay can be a standard for monitoring the levels of free DNA in recombinant proteins and viruses earmarked for commercial production of human therapeutics. Acknowledgments. The authors thank Mr. Andres Frei, Dr. Vijay Singh, Dr. Ronald Bordens, and Dr. Angel Cruz for encouragement and valuable suggestions and comments. This research was supported by funds granted by the Schering-Plough Research Institute.
REFERENCES 1. Henry, L. J., Xia, D., Wilke, M. E., Deisenhofer, J., and Gerard, R. D. (1994) J. Virol. 68, 5239–5246. 2. Philipson, L., and Lindberg, U. (1974) in Comprehensive Virology (Frankel-Conrat, H., and Wagner, R. R., Eds.), Vol. 3, pp. 143–227, Plenum, New York. 3. Cesarone, C. F., Bolognesi, C., and Santi, L. (1979) Anal. Biochem. 100, 188–197. 4. Labarca, C., and Paigen, K. (1980) Anal. Biochem. 102, 344– 352. 5. Huyghe, B. G., Liu, X., Sutjipto, S., Sugarman, B. J., Horn, M. T., Shepard, H. M., Scandella, C. J., and Shabram, P. (1995) Hum. Gene Ther. 6, 1403–1416. 6. Maizel, J. V., Jr., White, D. O., and Scharff, M. D. (1968) Virology 36, 115–125. 7. Green, M., and Pinˇa, M. (1963) Virology 20, 199–207. 8. Gallagher, S., and Rymaszewski, Z. (1994) Methods Toxicol. 1B, 164–177. 9. Loontiens, F. G., Regenfuss, P., Zechel, A., Dumortier, L., and Clagg, R. M. (1990) Biochemistry 29, 9029–9039. 10. Tibbetts, C., and Giam, C.-Z. (1979) J. Virol. 32(3), 995–1005. 11. Horwitz, M. S. (1990) in Virology (Fields, B. N., Knipe, D. M., et al., Eds.), 2nd ed., pp. 1679–1721, Raven Press, New York.
Targeted Deletion of a GA Tract by S1 Nuclease Benoit Barbeau and Eric Rassart1 De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, Montre´al, Que´bec, Canada H3C 3P8 Received October 18, 1996
Purine/pyrimidine sequences are abundant in eukaryotic genomic DNA. Certain of these DNA regions contain purine/pyrimidine mirror repeats and can show sensitity to S1 nuclease. Such regions are strongly indicative for the presence of a DNA structure known as H-DNA. This latter structure is characterized by the formation of a triple-stranded helix and a S1 nuclease1 To whom correspondence should be addressed at De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, C.P.8888 Succ. Centre-ville, Montre´al, PQ, Canada H3C 3P8. Fax: (514) 9874647.
ANALYTICAL BIOCHEMISTRY 247, 445–447 (1997) ARTICLE NO. AB972085
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
04-15-97 13:03:18
abnt
446
NOTES & TIPS
FIG. 1. Plasmid pSH2-7CAT compared to the deleted mutants. (A) 1500 bp of 5* flanking sequence was positioned upstream of the CAT gene (indicated by an arrow) (7) (GenBank Accession No. L47616). The 133-nt purine-rich segment is highlighted by a dark rectangle in the flanking region and the 24 GA repeats are presented below. The oligonucleotide B14 (small arrow) is positioned at the 5* end of the CAT gene in an inverted orientation. Relative distance of the presented map is indicated for the size of 200 bp. Both restriction sites BamHI and SacI are shown above the map. (B) S1 nuclease-treated clones 10S1-5, 50S1-1, and 10S1-4 were selected and sequenced using the oligonucleotide B14. Part of the purine-rich region is presented and includes the GA repeat. Deleted nucleotides for each mutant plasmids are indicated by a dash. Positioning of the deleted region is based on sequence comparison with the parental pSH2-7CAT plasmid.
sensitive single-stranded DNA segment. The formation of H-DNA is dependent on parameters such as pH, DNA supercoiling, and ion concentration (1–3). Although present in many different genomic regions, HDNA has been observed in several promoters, especially those categorized as TATA-less promoters (4, 5). Furthermore, it has been reported that H-DNA could block transcription (6). In order to assess the potential role of GA repeats (and thus potential H-DNA structure) in gene regulation, we have developed a protocol of targeted deletion of GA repeat structure using S1 nuclease, thus taking advantage of the presence of the potential H-DNA single-stranded region located in this segment. The plasmid used in this set of experiments is pSH2-7CAT and contains 1500 bp of the human 5* flanking region of the Fli-1 locus upstream of the CAT gene. A conserved GA repeat region made of 24 repeats lies near the 3* end of the 5* flanking region. This GA motif is present in a very purine-rich region (93%) of 133 bp (Fig. 1A). We have previously shown that the corresponding region of the mouse Fli-1 promoter region is S1 nuclease sensitive and thus suggestively adopts an H-DNA conformation (7). Circular pSH2-7 CAT plasmid (1 mg) was first treated with S1 nuclease following a modified version of the protocol of Johnson et al. (8). S1 nuclease treatment was performed for 25 min at 427C with increasing amounts of S1 nuclease (10, 20, 50, 100, and 200 units) (Pharmacia, Montreal, Canada) in 30 ml of 30 mM
NaOAc, pH 4.5, 300 mM NaCl, 0.2 mM EDTA, and 3 mM ZnCl2 . DNA samples were then extracted once with phenol–chloroform and separated with a 1% agarose gel. DNA fragments corresponding to linearized plasmids were extracted from the gel by filtering of the agarose plug through a Whatman paper to be finally ethanol precipitated. Next, the purified DNA was treated with 6 units of Klenow DNA polymerase (Pharmacia) in 20 ml 11 One-Phor-All buffer containing 250 mM dNTP for 30 min at 377C. After heat inactivation of the enzyme, 2 ml of the treated DNA was used for self-ligation in a final volume of 20 ml ligase buffer 11 (BRL) with 1 U T4 DNA ligase (Gibco BRL, Burlington, Canada) for 16 h at room temperature. Five microliters of the ligated product was used for DH5-a transformation. Bacterial clones containing high-molecularweight plasmid DNA were then selected. Sequence analysis was performed with the T7 Pharmacia sequencing kit (Pharmacia) and the oligonucleotide B14 (5*-GTGGTATATCCAGTGATTTT-3*) localized 40 bp from the 5* end of the CAT gene in the antisense strand (Fig. 1A). Of 20 bacterial clones, 3 were further selected for their plasmid DNA which showed targeted deletion of 12 to 13 GA repeats (clones 10S1-4 and 10S1-5 generated after a 10 U S1 treatment and clone 50S11 generated after a 50 U S1 treatment; Fig. 1B). Indeed, sequence analysis indicated that clones 10S15 and 10S1-4 had a deletion of the 5* half of the GA motif and included 7 and 28 bp of 5* flanking
AID
abnt
AB $NAT
/
6m2f$$$561
04-15-97 13:03:18
NOTES & TIPS
447
FIG. 2. Comparison of S1 nuclease sensitivity between pSH2-7CAT and deleted mutant plasmids. The parental plasmid pSH2-7CAT (A) and the deleted clones 10S1-4 (B) and 50S1-1 (C) were treated with different amounts of S1 nuclease (lane 2, 10 U; lane 3, 20 U; lane 4, 50 U; lane 5, 100 U; lane 6, 200 U) and separated with a 1% agarose gel. Comparison of S1 nuclease treatment can be made with the untreated sample (ND) for each plasmids. A linearized BamHI-digested pSH2-7CAT plasmid is also included in A. (R, relaxed; L, linear; S, supercoiled).
sequence, respectively (Fig. 1B). On the other hand, clone 50S1-1 had a deletion of the 3 * half of the GA motif and only included 1 bp downstream of the GA repeat (Fig. 1B). The two different types of GA repeat deletions (5* end and 3 * end) could be reminiscent of the presence of two different H-DNA isomers for the plasmid pSH2-7CAT. The specificity of the deletion was further confirmed by comparison of the restriction maps of the deleted and the wild-type plasmids, which showed no difference outside of the targeted region; in addition, no other apparent deletion in the surrounding sequences could be observed (data not shown). However, it should be emphasized that other types of non-B-DNA structures have not been investigated in this study and thus we cannot rule out that such other structures might also be sensitive to the described deletion protocol. To evaluate whether the pSH2-7CAT-derived mutants were less susceptible to S1 treatment, clones 10S1-4, 50S1-1, and the parental plasmid pSH27CAT were again treated with increasing amounts of S1 nuclease (Fig. 2). When comparisons of the intensity ratio between linear and relaxed forms were performed, plasmid pSH2-7CAT treated with 10 U S1 nuclease could be matched with clones 10S1-4 and 50S1-1 treated with 100 U and 50 U S1 nuclease, respectively (Fig. 2). These results thus demonstrate that the removal of GA repeat seems to have greatly reduced or even possibly abolished S1 sensitivity of this region in the pSH2-7 CAT plasmid and most likely renders the region less prone to form an HDNA structure. Although the actual mode by which complete S1 nuclease DNA cleavage is achieved has not been clarified in this study, several possibilities can be suggested including the targeting of the unpaired single-stranded region of H-DNA structure as well as the previously proposed S1 nuclease sensitivity of the Hoogsteen base
AID
AB $NAT
/
6m2f$$$562
04-15-97 13:03:18
pair in the triple-stranded helix (9). Several methods of deletion have been described and include exonuclease treatment (for example, BAL31, DNaseI, or exoIII) (10) and PCR-based deletion protocols (11). Unlike these other techniques, our method seemingly targets region of potential H-DNA structures and could thus help deciphering the role played by these latter in different promoters. Acknowledgments. We gratefully acknowledge Richard Bergeron for excellent technical assistance. B.B. was supported by a Quebec FCAR scholarship. This work was supported by the Medical Research Council of Canada (Grant MT7754).
REFERENCES 1. Hanvey, J. C., Shimizu, M., and Wells, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 6292–6295. 2. Kohwi, Y., and Kohwi-Shigematsu, T. (1993) J. Mol. Biol. 231, 1090–1101. 3. Mirkin, S. M., and Frank-Kamenetskii, M. D. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 541–576. 4. Firulli, A. B., Maibenco, D. C., and Kinniburgh, A. J. (1992) Biochem. Biophys. Res. Commun. 185, 264–270. 5. Pestov, D. G., Dayn, A., Siyanova, E. Y., George, D. L., and Mirkin, S. M. (1991) Nucleic Acids Res. 19, 6527–5632. 6. Grabczyk, E., and Fishman, M. C. (1995) J. Biol. Chem. 270, 1791–1797. 7. Barbeau, B., Bergeron, D., Beaulieu, M., Nadjem, Z., and Rassart, E. (1996) Biochim. Biophys. Acta 1307, 220–232. 8. Johnson, A. C., Jinno, Y., and Merlino, G. T. (1988) Mol. Cell Biol. 8, 4174–4184. 9. Pulleyblank, D. E., Haniford, D. B., and Morgan, R. (1985) Cell 42, 271–280. 10. Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual. 2th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 11. Vallette, F., Mege, E., Reiss, A., and Adesnik, M. (1989) Nucleic Acids Res. 17, 723–733.
abnt