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Properties and regulation of the UVRABC endonuclease
BIOCH1MIE, 1982, 64, 595-598.
CNRS symposium, May 1982 - TOULOUSE
Inducible responses to D N A damages
Properties and regulation of the UVRABC endonuclease. W. Dean RUPP <>, Aziz SANCAR and Gwendotyn B. SANCAR.
Departments of Therapeutic Radiology and Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven - CT 06510 USA.
Rdsumd.
Summary.
Ce rapport rdsume le clonage des gknes uvrA, uvrB et uvrC d'E. coli, l'identification et l'isolement des produits des gknes, la rdgulation des gbnes et la reconstitution d'une endonucldase U V R A B C ?~ partir des composants isolds individuellement.
This report summarizes the cloning of the uvrA, uvrB and uvrC genes of E. coli, the identification and isolation of the gene products, the regulation of the genes, and reconstitution of active U V R A B C endonuclease from the individually i~olated components.
Mots-el~s : uvrA, uvrB, uvrC d'E. eoli / endonuei~ase UVRABC / r6paration du DNA.
Key-words: E. eoU uvrA, uvrB, uvrC / UVRABC endonuelease / DNA repair.
Introduction.
While it has been clear since the early 1960's that the uvrA, uvrB and uvrC genes are essential for an early step in nucleotide excision repair, progress in purifying and characterizing the UVRABC enzyme has proved to be very difficult because of the low activity and instability of the enzyme. Several developments have contributed to a rapid increase in our understanding of these proteins and the nucleotide excision mechanism. On the one hand, certain experiments emphasized significant differences between the reaction catalyzed by the UVRABC enzyme and the smaller and simpler UV endonucleases from M. luteus and T4 bacteriophage that specifically recognize pyrimidine dimers and which are now known to have combined glycosylase and AP endonuclease activities (see ref. 1 for summary). The UVRABC enzyme required ATP and Mg ~÷ for its intracellular activity while the other purified enzymes did not. Seeberg and his colleagues [2-5] also showed that products of the 3 uvr genes were each required for the incision step and that they were easily dissociated during purification thus explaining the great difficulty in obtaining highly purified active enzyme. On the other hand, other laboratories began using DNA recombinant methodology for cloning the genes with the expectation that this approach would lead initially to the identification of the gene products <>To whom all correspondence should be addressed.
and then to their production in increased amounts to facilitate the biochemical characterization of the UVRABC enzyme and its activities. This report will concentrate on results obtained from the latter approach. Cloning of the uvr genes.
Several laboratories have been active in cloning and characterizing the uvrA, uvrB and uvrC genes [6-19]. The uvrA gene was cloned directly from the E. coli chromosome [7, 8]. The uvrB gene was located on a plasmid in the Clarke-Carbon collection and was then subcloned into pBR322 [6, 9]. In another approach [13], the uvrB gene from a lambda transducing phage was subcloned. Van Sluis and Pannekoek [14] found a plasmid in the Clarke-Carbon collection and subcloned uvrC from it on a 3.4 kb Pst I fragment. Our laboratory subsequently derived a similar uvrC plasmid carrying the same 3.4 kb Pst I fragment [10]. Restriction mapping [20] and sequence analysis (Sancar, Sancar and Rupp, unpublished data) demonstrated that one of the ends was in the chromosomal DNA while the Pst I site at the other end came from the ColE1 vector. Yoakum et al. ,[17] reported cloning the uvrC gene directly from chromosomal DNA on a Pst I fragment of 3.4 kb, a result that is inconsistent with the restriction mapping data of Ohta et al. [2t)] which showed that the uvrC gene is on a chromosomal Pst I fragment longer than 4.6 kb.
596
W. D. R u p p and coll.
Identification of the gene products. When a cloned gene is obtained, the next step is to determine the identity of the gene product. Several methods were developed and adapted in our laboratory to make this task easier. The maxicell procedure was used to specifically label those proteins made by multicopy plasmids, including any proteins encoded by DNA cloned into such plasmids [21]. While this reduced the number of candidates for the gene products to a small number, a further refinement was necessary for the unambiguous assignment of a protein product to a particular cloned gene. The insertion of a transposon in a gene was the key that allowed such assignments to be made [7, 8]. Insertions in genes are detected genetically by the inactivation of a gene, and the locations of the inserts are determined physically by mapping the mutated plasmids with restriction enzymes. Since the mutated genes do not make normal gene products, the disappearance of a specific protein in maxicells meant that it was the product of the mutated gene. Using this approach, we have identified the products of the uvrA, uvrB and uvrC genes as polypeptides of 114 kd, 84 kd and 70 kd [8-10]. Using this methodology, other laboratories have also identified the uvrC gene product [18, 19]. Orientation of genes and location of control regions. Additional information obtained during the transposon inactivation of the cloned genes was used to determine the boundaries of the cloned gene and also its orientation. In many cases, insertion of the transposon in a structural gene resulted in the synthesis of truncated polypeptides that result from termination signals in the inserted transposon near the site of insertion. Thus the length of the truncated products corresponds with the position of the transposon insertion so that the beginning and the end of the structural gene can both be defined within about a hundred base pairs. This precision is sufficient to isolate the appropriate restriction fragments carrying the gene's promoter and control regions in those situations when they are adjacent to the structural gene and not separated from it. This approach was used to locate and sequence the promoters and control regions for uvrA [8, 21], uvrB [9, 22] and uvrC [10, 23]. Regulation of the uvr genes. The regulation of the uvr genes is more intricate than had been anticipated. Results of Kenyon and Walker [2A] indicated that uvrA is under control of recA and lexA while experiments of Fogliano BIOCH1MIE, 1982, 64, n° 8-9.
and Schendel [25] indicated that uvrB is also similarly controlled. Direct measurement of uvrA complementing activity and UVRA protein confirmed these results [26] while similar experiments with uvrB (unpublished data) were inconclusive. Sequencing of the control regions of uvrA [21] and uvrB [22, 27] showed the presence of a sequence similar to the LEXA-binding sequences (SOS boxes) previously described by Little and Mount [28], Brent and Ptashne [29], and Horii et al. [30] for the lexA and recA genes. Footprinting experiments showed that LEXA does bind to these sites in uvrA [21] and uvrB [22] protecting them against nuclease digestion, and transcription experiments with purified RNA polymerase showed that the binding of LEXA prevented transcription from those promoters with LEXA binding sites [21, 22]. While the uvrA control region is simple with a single promoter controlled by a LEXA binding site [21], the uvrB control region is more complex and contains multiple promoters [22, 27]. Promoter P1 (the one closest to the uvrB structural gene) is unaffected by LEXA while promoter P2 contains a LEXA binding site and is under LEXA regulation. A third promoter, P3, is about 300 bp upstream from P2 and does not have a LEXA binding site but is nevertheless regulated by binding of LEXA at P2. It is not certain whether P3 has an intracellular role in the expression of uvrB because the in vitro transcript from P3 terminates in the middle of the P2 LEXA binding site even in the absence of LEXA [22]. However, it is possible that in the intact cell other factors might alleviate this termination and allow transcripts from P3 to extend through the uvrB structural gene. The extent of the uvrC gene has been defined by deletion analysis of a plasmid carrying the cloned gene and the results indicate that the promoter is adjacent to the 5' end of the uvrC structural gene (Sancar, Sancar and Rupp, unpublished observations), in contrast to a report claiming that the uvrC gene is under the control of a distant promoter [19]. The promoter region of the uvrC gene does not contain a sequence obviously similar to other LEXA binding sites. Thus, the data available now suggest that uvrC is not directly regulated by recA and lexA. Since little is known about the stoichiometry of the various components in the active UVRABC endonuclease, it is not possible to predict what effect there would be on enzyme activity if one or two of the components were increased after irradiation while the third remained constant. If the
UVRABC
rate-limiting component does not increase, then the level of enzyme activity is expected to remain unchanged. It also remains possible that additional loops of control exist for regulation. For example the sequence A A T T T G T G T C A T A A T T A A found in the uvrA promoter resembles the sequence AATTTGTTGGCATAATTAA in uvrB that extends from P2 into P1 [21]. The extensive conservation of this sequence strongly suggests that its presence in the control regions of both uvrA and uvrB is not fortuitous. For example, it would be expected that a protein binding specifically to this sequence would be a repressor for uvrA and for both promoters P1 and P2 of uvrB in contrast to L E X A which represses only P2. Construction o[ plasmids that overproduce the uvr gene products.
Introducing the uvrA plasmid into a strain with defective L E X A resulted in about a hundred fold increase in the level of U V R A making it reasonable to use such cells for the convenient preparation of U V R A ,[26]. A similar approach for uvrB and uvrC did not result in a similar level of overproduction so the more direct approach of subcloning the genes into vectors favorable for overexpression was taken. Two different vectors were used. The tac promoter vector was obtained from A m a n n and Brosious. This is a hybrid promoter containing the - - 3 5 region of the trp promoter anc~ the Pribnow box of the lac promoter. Both of these match exactly the canonical sequences so the hybrid promoter should be ideal. In addition, the tac promoter retains the control by the lac repressor so that overproduction can be turned off to prevent killing of the host cell. The uvrA and uvrC genes have been hooked up to this promoter. When induced, cells containing the plasmid with the uvrA gene make far more U V R A than any other protein in the cell, while induced cells containing the uvrC plasmid make amounts of U V R C comparable to other major proteins in the cell. The uvrB gene was cloned into a vector whose replication is uncontrolled at high temperature [31]. Growing cells with this plasmid at high temperature results in the synthesis of U V R B in quantities greater than any other cellular protein. With the availability of these plasmids, it is now feasible to easily obtain the three U V R A B C components in useful quantities at high purity. Activities of the U V R A B C and its components.
endonuclease
The three components have been mixed to form active U V R A B C endonuclease that acts on D N A ' s BIOCHIMIE, 198'2, 64, n ° 8-9.
597
endonuclease.
damaged with UV, hydroxymethyl-psoralen plus near U V and cis-platinum [32]. All three purified proteins are required in that the individual proteins or mixtures of any 2 of them do not result in cutting of damaged DNA. Sequencing gels have been used to define the location of the cuts made in UV-irradiated D N A and the results will be reported in detail [32]. In summary, the activity of this enzyme is extremely novel in that cuts are made on both sides of U V lesions (including pyrimidine-C adducts as well as normal cyclobutane pyrimidine dimers) in the damaged D N A strand 12 nucleotides apart including 7 undamaged nucleotides on the 5' side of the lesion and 3 undamaged nucleotides on the 3' side. It thus seems clear that the classical models for nucleotide excision repair based on nicking in the single-stranded distorted region followed by excision mediated by the 5' to 3' nuclease of D N A polymerase I must be revised.
Acknowledgements. This work was supported by United Public Health Service Grant CA 06519 and American Cancer Society Grant PDT 80.
REFERENCES. 1. Seeberg, E. (1981) Prog. Nucl. Acid. Res. & Molec. Biol., 26, 217-226. 2. Seeberg, E., Nissen-Meyer, J. & Strike, P. (1976) Nature, 263, 524-526. 3. Seeberg, E. (1978) Proc. Natl. Acad. Sci. USA, 75, 2569-2573. 4. Seeberg, E. (1978) in <> (P. C. Hanawal't, E. C. Friedberg and C. F. Fox, eds.), Academic Press, New York, pp. 229-235. 7. SaBcar, A. & l~upp, W. D. (1979) Biochem. Biophys. Res. Comm., 90, 123-129. 8. Sancar, A., Whaz'to~, R. P., Seltzer, S., Kacinski, B. M., Clarke, N. D. & Rupp, W. D. (1981) J. Mol. Biol., 148, 45-62. 9. Sancar, A., Clarke, I'4. D., Griswold, J., Kennedy, W. J. & RBpp, W. D. (1981) J. Mol. Biol., 148, 63-70. 10. Sancar, A., Ka~inski, B. M., Mott, D. L. & Rupp, W. D. (1981) Proc. Natl. Aead. Sci. USA, 78, 5450-5454. 11. Sancar, A., Williams, K. R., Chase, J. W. & Rupp, W. D. (1981) Proc. Natl. Acad. Sci. USA, 78, 4274-4278. 12. Auerbach, J. & Howard-Flanders, P. (1979) Molec. Gen. Genet., 1'68, 341-344. 13. Pannekoek, H., Noordemeex, I. A., van Sluis, C. A. & van de Putt~, P. (1978) I. Bacteriol., 135, 884896.
598
W . D. R u p p and coll.
14. van Sluis, C. A. & Paanekoek, H. (1978) J. Supramolec. Struct., Suppl. 2, 51. 15. Brartdsma, J. A., van Sluis, C. A. & van de Putte, P. (1981) J. Bacteriol., 147, 682-684. 16. Seeberg, E., Steiaum, A. L. & Blingsmo, O. R. (1978) 1. Supramolec. Struct., Suppl. 2, 51. 17. Yoakum, G. H., K,ushner, S. R. & Grossman, L. (1981) Gene, 12, 243-248. 18. Yoakum, G. H. & Grossman, L. (1981) Nature, 292, 171-173. 19. Sharma, S., Ohta, A., Dowhan, W. & Moses, R. E. (1981) Proc. Natl. Acad. Sci. USA, 78, 6033-6037. 20. Ohta, A., Waggoner, K., Radominska-Pyrek, A. Dowhan, W. (1981) J. Bacteriol., 147, 552-562. 21. Sancar, A., Sartcar, G. B., Rupp, W. D., Little, J. W. & Mount, D. W. (1982) Nature, 298, 96-98. 22. San'car, G. B., Sanear, A., Little, J. W. and Rupp, W. D. (1982) Cell, 28, 523-530. 23. Sancar, G. B., Sancar, A. & Rupp. W. D., unpublished data.
BIOCHIMIE, 1982, 64, n ° 8-9.
24. Kenyon, C. ,~ Walker, G. (1981) Nature, 289, 808810. 25. Fogliano, M. & Sehendel, P. (1981) Nature, 289, 196198. 26. Kacinski, B. M., Sanear, A. ~ Rupp, W. D. (1981) Nucleic Acids Res., 9, 4495-4508. 27. van den Berg, E., Zwersloot, J., Noordemeer, I., Pannekoek, H., Dekker, B., Dijkema, R. & van Ormondt, H. (1981) Nucleic Acids Res., 9, 56235643. 28. Little, J. W., Mount, D. W. & Yanisch-Perron, C. R. (1981) Proc. Natl. ,4cad. Sci. USA, 78, 4199-4203. 29. Brent, R. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 4199-4203. 29. Brent, R. • Ptaslme, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 4204-4281. 30. Horii, T., Ogawa, T. & Ogawa, H. (1981) Cell, 23, 689-697. 31. Bittner, M. • Vapnek, D. (1981) Gene, 15, 319-329. 32. Sancar, A. & Rupp, W. D. (1982) submitted for publication.
CNRS symposium, May 1982 - TOULOUSE
Inducible responses to D N A damages
Properties and regulation of the UVRABC endonuclease. W. Dean RUPP <>, Aziz SANCAR and Gwendotyn B. SANCAR.
Departments of Therapeutic Radiology and Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven - CT 06510 USA.
Rdsumd.
Summary.
Ce rapport rdsume le clonage des gknes uvrA, uvrB et uvrC d'E. coli, l'identification et l'isolement des produits des gknes, la rdgulation des gbnes et la reconstitution d'une endonucldase U V R A B C ?~ partir des composants isolds individuellement.
This report summarizes the cloning of the uvrA, uvrB and uvrC genes of E. coli, the identification and isolation of the gene products, the regulation of the genes, and reconstitution of active U V R A B C endonuclease from the individually i~olated components.
Mots-el~s : uvrA, uvrB, uvrC d'E. eoli / endonuei~ase UVRABC / r6paration du DNA.
Key-words: E. eoU uvrA, uvrB, uvrC / UVRABC endonuelease / DNA repair.
Introduction.
While it has been clear since the early 1960's that the uvrA, uvrB and uvrC genes are essential for an early step in nucleotide excision repair, progress in purifying and characterizing the UVRABC enzyme has proved to be very difficult because of the low activity and instability of the enzyme. Several developments have contributed to a rapid increase in our understanding of these proteins and the nucleotide excision mechanism. On the one hand, certain experiments emphasized significant differences between the reaction catalyzed by the UVRABC enzyme and the smaller and simpler UV endonucleases from M. luteus and T4 bacteriophage that specifically recognize pyrimidine dimers and which are now known to have combined glycosylase and AP endonuclease activities (see ref. 1 for summary). The UVRABC enzyme required ATP and Mg ~÷ for its intracellular activity while the other purified enzymes did not. Seeberg and his colleagues [2-5] also showed that products of the 3 uvr genes were each required for the incision step and that they were easily dissociated during purification thus explaining the great difficulty in obtaining highly purified active enzyme. On the other hand, other laboratories began using DNA recombinant methodology for cloning the genes with the expectation that this approach would lead initially to the identification of the gene products <>To whom all correspondence should be addressed.
and then to their production in increased amounts to facilitate the biochemical characterization of the UVRABC enzyme and its activities. This report will concentrate on results obtained from the latter approach. Cloning of the uvr genes.
Several laboratories have been active in cloning and characterizing the uvrA, uvrB and uvrC genes [6-19]. The uvrA gene was cloned directly from the E. coli chromosome [7, 8]. The uvrB gene was located on a plasmid in the Clarke-Carbon collection and was then subcloned into pBR322 [6, 9]. In another approach [13], the uvrB gene from a lambda transducing phage was subcloned. Van Sluis and Pannekoek [14] found a plasmid in the Clarke-Carbon collection and subcloned uvrC from it on a 3.4 kb Pst I fragment. Our laboratory subsequently derived a similar uvrC plasmid carrying the same 3.4 kb Pst I fragment [10]. Restriction mapping [20] and sequence analysis (Sancar, Sancar and Rupp, unpublished data) demonstrated that one of the ends was in the chromosomal DNA while the Pst I site at the other end came from the ColE1 vector. Yoakum et al. ,[17] reported cloning the uvrC gene directly from chromosomal DNA on a Pst I fragment of 3.4 kb, a result that is inconsistent with the restriction mapping data of Ohta et al. [2t)] which showed that the uvrC gene is on a chromosomal Pst I fragment longer than 4.6 kb.
596
W. D. R u p p and coll.
Identification of the gene products. When a cloned gene is obtained, the next step is to determine the identity of the gene product. Several methods were developed and adapted in our laboratory to make this task easier. The maxicell procedure was used to specifically label those proteins made by multicopy plasmids, including any proteins encoded by DNA cloned into such plasmids [21]. While this reduced the number of candidates for the gene products to a small number, a further refinement was necessary for the unambiguous assignment of a protein product to a particular cloned gene. The insertion of a transposon in a gene was the key that allowed such assignments to be made [7, 8]. Insertions in genes are detected genetically by the inactivation of a gene, and the locations of the inserts are determined physically by mapping the mutated plasmids with restriction enzymes. Since the mutated genes do not make normal gene products, the disappearance of a specific protein in maxicells meant that it was the product of the mutated gene. Using this approach, we have identified the products of the uvrA, uvrB and uvrC genes as polypeptides of 114 kd, 84 kd and 70 kd [8-10]. Using this methodology, other laboratories have also identified the uvrC gene product [18, 19]. Orientation of genes and location of control regions. Additional information obtained during the transposon inactivation of the cloned genes was used to determine the boundaries of the cloned gene and also its orientation. In many cases, insertion of the transposon in a structural gene resulted in the synthesis of truncated polypeptides that result from termination signals in the inserted transposon near the site of insertion. Thus the length of the truncated products corresponds with the position of the transposon insertion so that the beginning and the end of the structural gene can both be defined within about a hundred base pairs. This precision is sufficient to isolate the appropriate restriction fragments carrying the gene's promoter and control regions in those situations when they are adjacent to the structural gene and not separated from it. This approach was used to locate and sequence the promoters and control regions for uvrA [8, 21], uvrB [9, 22] and uvrC [10, 23]. Regulation of the uvr genes. The regulation of the uvr genes is more intricate than had been anticipated. Results of Kenyon and Walker [2A] indicated that uvrA is under control of recA and lexA while experiments of Fogliano BIOCH1MIE, 1982, 64, n° 8-9.
and Schendel [25] indicated that uvrB is also similarly controlled. Direct measurement of uvrA complementing activity and UVRA protein confirmed these results [26] while similar experiments with uvrB (unpublished data) were inconclusive. Sequencing of the control regions of uvrA [21] and uvrB [22, 27] showed the presence of a sequence similar to the LEXA-binding sequences (SOS boxes) previously described by Little and Mount [28], Brent and Ptashne [29], and Horii et al. [30] for the lexA and recA genes. Footprinting experiments showed that LEXA does bind to these sites in uvrA [21] and uvrB [22] protecting them against nuclease digestion, and transcription experiments with purified RNA polymerase showed that the binding of LEXA prevented transcription from those promoters with LEXA binding sites [21, 22]. While the uvrA control region is simple with a single promoter controlled by a LEXA binding site [21], the uvrB control region is more complex and contains multiple promoters [22, 27]. Promoter P1 (the one closest to the uvrB structural gene) is unaffected by LEXA while promoter P2 contains a LEXA binding site and is under LEXA regulation. A third promoter, P3, is about 300 bp upstream from P2 and does not have a LEXA binding site but is nevertheless regulated by binding of LEXA at P2. It is not certain whether P3 has an intracellular role in the expression of uvrB because the in vitro transcript from P3 terminates in the middle of the P2 LEXA binding site even in the absence of LEXA [22]. However, it is possible that in the intact cell other factors might alleviate this termination and allow transcripts from P3 to extend through the uvrB structural gene. The extent of the uvrC gene has been defined by deletion analysis of a plasmid carrying the cloned gene and the results indicate that the promoter is adjacent to the 5' end of the uvrC structural gene (Sancar, Sancar and Rupp, unpublished observations), in contrast to a report claiming that the uvrC gene is under the control of a distant promoter [19]. The promoter region of the uvrC gene does not contain a sequence obviously similar to other LEXA binding sites. Thus, the data available now suggest that uvrC is not directly regulated by recA and lexA. Since little is known about the stoichiometry of the various components in the active UVRABC endonuclease, it is not possible to predict what effect there would be on enzyme activity if one or two of the components were increased after irradiation while the third remained constant. If the
UVRABC
rate-limiting component does not increase, then the level of enzyme activity is expected to remain unchanged. It also remains possible that additional loops of control exist for regulation. For example the sequence A A T T T G T G T C A T A A T T A A found in the uvrA promoter resembles the sequence AATTTGTTGGCATAATTAA in uvrB that extends from P2 into P1 [21]. The extensive conservation of this sequence strongly suggests that its presence in the control regions of both uvrA and uvrB is not fortuitous. For example, it would be expected that a protein binding specifically to this sequence would be a repressor for uvrA and for both promoters P1 and P2 of uvrB in contrast to L E X A which represses only P2. Construction o[ plasmids that overproduce the uvr gene products.
Introducing the uvrA plasmid into a strain with defective L E X A resulted in about a hundred fold increase in the level of U V R A making it reasonable to use such cells for the convenient preparation of U V R A ,[26]. A similar approach for uvrB and uvrC did not result in a similar level of overproduction so the more direct approach of subcloning the genes into vectors favorable for overexpression was taken. Two different vectors were used. The tac promoter vector was obtained from A m a n n and Brosious. This is a hybrid promoter containing the - - 3 5 region of the trp promoter anc~ the Pribnow box of the lac promoter. Both of these match exactly the canonical sequences so the hybrid promoter should be ideal. In addition, the tac promoter retains the control by the lac repressor so that overproduction can be turned off to prevent killing of the host cell. The uvrA and uvrC genes have been hooked up to this promoter. When induced, cells containing the plasmid with the uvrA gene make far more U V R A than any other protein in the cell, while induced cells containing the uvrC plasmid make amounts of U V R C comparable to other major proteins in the cell. The uvrB gene was cloned into a vector whose replication is uncontrolled at high temperature [31]. Growing cells with this plasmid at high temperature results in the synthesis of U V R B in quantities greater than any other cellular protein. With the availability of these plasmids, it is now feasible to easily obtain the three U V R A B C components in useful quantities at high purity. Activities of the U V R A B C and its components.
endonuclease
The three components have been mixed to form active U V R A B C endonuclease that acts on D N A ' s BIOCHIMIE, 198'2, 64, n ° 8-9.
597
endonuclease.
damaged with UV, hydroxymethyl-psoralen plus near U V and cis-platinum [32]. All three purified proteins are required in that the individual proteins or mixtures of any 2 of them do not result in cutting of damaged DNA. Sequencing gels have been used to define the location of the cuts made in UV-irradiated D N A and the results will be reported in detail [32]. In summary, the activity of this enzyme is extremely novel in that cuts are made on both sides of U V lesions (including pyrimidine-C adducts as well as normal cyclobutane pyrimidine dimers) in the damaged D N A strand 12 nucleotides apart including 7 undamaged nucleotides on the 5' side of the lesion and 3 undamaged nucleotides on the 3' side. It thus seems clear that the classical models for nucleotide excision repair based on nicking in the single-stranded distorted region followed by excision mediated by the 5' to 3' nuclease of D N A polymerase I must be revised.
Acknowledgements. This work was supported by United Public Health Service Grant CA 06519 and American Cancer Society Grant PDT 80.
REFERENCES. 1. Seeberg, E. (1981) Prog. Nucl. Acid. Res. & Molec. Biol., 26, 217-226. 2. Seeberg, E., Nissen-Meyer, J. & Strike, P. (1976) Nature, 263, 524-526. 3. Seeberg, E. (1978) Proc. Natl. Acad. Sci. USA, 75, 2569-2573. 4. Seeberg, E. (1978) in <
598
W . D. R u p p and coll.
14. van Sluis, C. A. & Paanekoek, H. (1978) J. Supramolec. Struct., Suppl. 2, 51. 15. Brartdsma, J. A., van Sluis, C. A. & van de Putte, P. (1981) J. Bacteriol., 147, 682-684. 16. Seeberg, E., Steiaum, A. L. & Blingsmo, O. R. (1978) 1. Supramolec. Struct., Suppl. 2, 51. 17. Yoakum, G. H., K,ushner, S. R. & Grossman, L. (1981) Gene, 12, 243-248. 18. Yoakum, G. H. & Grossman, L. (1981) Nature, 292, 171-173. 19. Sharma, S., Ohta, A., Dowhan, W. & Moses, R. E. (1981) Proc. Natl. Acad. Sci. USA, 78, 6033-6037. 20. Ohta, A., Waggoner, K., Radominska-Pyrek, A. Dowhan, W. (1981) J. Bacteriol., 147, 552-562. 21. Sancar, A., Sartcar, G. B., Rupp, W. D., Little, J. W. & Mount, D. W. (1982) Nature, 298, 96-98. 22. San'car, G. B., Sanear, A., Little, J. W. and Rupp, W. D. (1982) Cell, 28, 523-530. 23. Sancar, G. B., Sancar, A. & Rupp. W. D., unpublished data.
BIOCHIMIE, 1982, 64, n ° 8-9.
24. Kenyon, C. ,~ Walker, G. (1981) Nature, 289, 808810. 25. Fogliano, M. & Sehendel, P. (1981) Nature, 289, 196198. 26. Kacinski, B. M., Sanear, A. ~ Rupp, W. D. (1981) Nucleic Acids Res., 9, 4495-4508. 27. van den Berg, E., Zwersloot, J., Noordemeer, I., Pannekoek, H., Dekker, B., Dijkema, R. & van Ormondt, H. (1981) Nucleic Acids Res., 9, 56235643. 28. Little, J. W., Mount, D. W. & Yanisch-Perron, C. R. (1981) Proc. Natl. ,4cad. Sci. USA, 78, 4199-4203. 29. Brent, R. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 4199-4203. 29. Brent, R. • Ptaslme, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 4204-4281. 30. Horii, T., Ogawa, T. & Ogawa, H. (1981) Cell, 23, 689-697. 31. Bittner, M. • Vapnek, D. (1981) Gene, 15, 319-329. 32. Sancar, A. & Rupp, W. D. (1982) submitted for publication.