Aurora-A Abrogation of p53 DNA Binding and Transactivation Activity by Phosphorylation of Serine 215

Abstract

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164–171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and γ-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55–62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function. The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164–171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and γ-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55–62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function. Aurora is a subfamily of serine/threonine protein kinases. In vertebrates, they comprise Aurora-A, -B, and -C, with one or more highly conserved orthologues being found in the yeasts, flies, worms, and other invertebrates. Saccharomyces cerevisiae have a Aurora W. Biol. The and one in of the Aurora-A and R. Curr. Opin. Genet. The of Aurora-A and have been found in R. that Aurora a in progression of the cell to cell cycle such as and and and R. Curr. Opin. Genet. the that the is in been that of the Aurora-A in as as of and S. of Aurora-A in as as and S. have that Aurora-A important in cell and S. S. However, the molecular mechanism that Aurora-A p53 by phosphorylation of in S. S. Nat. Genet. we that DNA binding and transactivation activity of p53 was by Aurora-A p53 at Ser-215 in and in The of p53 DNA binding and transactivation activity by Aurora-A on phosphorylation of Ser-215 not Aurora-A phosphorylation of Ser-215 of p53 is with cell cycle cell and data indicate that p53 is a of Aurora-A and that Aurora-A its function phosphorylation of Ser-215 of In Ser-215 as a phosphorylation site of p53 that by other serine/threonine and p53 protein was were of p53 and Aurora-A were by of the of p53 and Aurora-A and of p53 were with a as a and in and were as Biol. In kinase was with in a or p53 as The was at for and in of p53 was by and were in and with or and in the to and a were and and were to the activity was by the activity to the activity. was of p53 transactivation and DNA binding activity on phosphorylation of Ser-215 not were with and with and and and were with of were to and as The was with in the of and for the of p53 binding to and show the of and the and of p53, p53 was and the by in a and for and with a of the was to by with for at The was with of or for at by at in the of a of the and were as Biol. the the were by on a of The was for at in for the and for at in for the were with were by with in of p53 were by for at in The was by in The were by for at and the with The were to as to the phosphorylation sites Biol. were to and to of the the was with to and the and the was by was as S. Z. Biol. was with the p53 DNA binding site at for for p53 binding activity was in the of a of the or and of was by the of a for p53 The were by a and cell cycle were in in and with was by were with and a to the The of apoptosis was by was as were with p53 and Aurora-A or The was a p53 at Aurora-A with tumor suppressor p53 Aurora-A and p53 to and the of Aurora-A and of p53 function in of and R. S. S. Nat. Genet. Aurora-A with p53 that Aurora-A phosphorylated p53 in and in that Aurora-A phosphorylated p53 on serine p53 phosphorylated by Aurora-A, p53 was with and the were to by of the that the activity of was the cycle of the in the the not a the of p53, the that serine or at in its is phosphorylated serine indicate that Ser-215 is a phosphorylation site of p53 for Aurora-A result, p53 was with and and that the was Ser-215 and the other was and the of Ser-215 to Aurora-A phosphorylation of p53 in and in and Ser-215 of p53 is a major by p53 at of Aurora-A with cell were with and with and Aurora-A phosphorylation of p53 in and in were with of the were with The were to in kinase p53 as a The is in were with with or with in for and with The were by to to the and by of Aurora-A was with Aurora-A phosphorylation of p53 on serine The phosphorylated p53 protein by Aurora-A was in kinase and by as and p53 was with or and the were to of for cycle is in the been to at the R. in and the p53 that a serine in cycle of The a was and with that the that either or Aurora-A phosphorylation of p53 and the and in Aurora-A kinase and in were as p53-S215A as a p53 at of Aurora-A with cell were with and with and Aurora-A phosphorylation of p53 in and in were with of the were with The were to in kinase p53 as a The is in were with with or with in for and with The were by to to the and by of Aurora-A was with Aurora-A phosphorylation of p53 on serine The phosphorylated p53 protein by Aurora-A was in kinase and by as and p53 was with or and the were to of for cycle is in the been to at the R. in and the p53 that a serine in cycle of The a was and with that the that either or Aurora-A phosphorylation of p53 and the and in Aurora-A kinase and in were as p53-S215A as a p53 DNA and Ser-215 of p53, we Aurora-A DNA binding and transactivation activity of p53 that p53 DNA binding activity was inhibited by of Aurora-A in and of Aurora-A by the DNA binding activity The effect of Aurora-A on p53 transactivation activity was with p21Cip/WAF1 and in Aurora-A inhibited and activity in a the of Aurora-A by the activity of and were by of p53 and Aurora-A in protein of and were by of Aurora-A and by of Aurora-A data indicate that Aurora-A p53 and its DNA binding and transactivation of p53 and DNA on of Ser-215 phosphorylation of Ser-215 is for Aurora-A of p53 DNA binding and transactivation activity. Aurora-A phosphomimic p53-S215D and p53-S215A were by Ser-215 to and respectively. were in and that p53-S215A induced the and activity of and PTEN, to of However, of p53, were not inhibited by of phosphomimic p53-S215D lost the to induce and and activity. In p53-S215A retained DNA binding was not inhibited by Aurora-A, p53-S215D to to DNA we that Aurora-A of p53 on phosphorylation of that Aurora-A p53 at Ser-315, was in and to of p53 S. S. Nat. Genet. However, the its on p53 DNA binding and transactivation activity we phosphomimic as as and and that phosphomimic and retained p53 DNA binding and transactivation activity and were by Aurora-A and However, and to DNA binding data indicate that Aurora-A p53 phosphorylation of Ser-215 not of p53 on of p53 is a tumor suppressor and a of cell and cell cycle we the of Aurora-A phosphorylation of Ser-215 on p53 function. and have that to apoptosis and that p53 the response of the cell to cisplatin Biol. we the effect of Aurora-A phosphorylation of p53 on cell in The were with of p53, with and with and that of p53-S215A or p53 cisplatin and induced apoptosis at However, of Aurora-A inhibited the apoptosis induced by p53 not of apoptosis was in the and that were by In p53-S215D lost the to to cell p53, and not p53-S215D inhibited in and the cell Aurora-A and effect on p53-S215A In that the cell cycle at in p53, and were by Aurora-A and of Aurora-A, to the cell cycle arrest in with arrest was not in p53-S215D in response to is that of p53-S215D on cell and cell cycle progression as with with and data indicate that phosphorylation of Ser-215 not of p53 by Aurora-A a effect on p53 of p53 function phosphorylation of were with of p53 and with or of were with cisplatin for was by in and were with the of was was in and cell cycle with p53, Aurora-A, and were with at Cell cycle was in of p53 function phosphorylation of were with of p53 and with or of were with cisplatin for was by in and were with the of was was in and cell cycle with p53, Aurora-A, and were with at Cell cycle was in have that Aurora-A proteins that important for proteins a in of the to mitotic C. S. Biol. for its in of a protein for and of R. S. C. Cell Biol. a protein in and and stability S. Biol. and is to R. Cell Biol. In Ser-215 of p53 been identified as a phosphorylation site of Aurora-A phosphorylation of p53 p53 DNA binding and transactivation activity. As a result, p53 target such as and PTEN, were by function and cell cycle arrest in response to DNA were by important for they provide a of the Aurora-A function in cell cell and a Aurora-A and the DNA been is the of a phosphorylation site p53 is of the that its activity as a transactivation the conserved a and a been that phosphorylation of p53 in of the of to p53 in the and of the of p53, to its phosphorylation of p53 the at or can the or protein B. R. S. S. Biol. phosphorylation of p53 protein at sites DNA or can the of C. the of p53 in the is a posttranslational important in the activity of p53 in of the DNA binding function of p53 in and in the of the of not of been that is a of and C. S. C. of p53 at site the DNA binding activity of p53 in C. B. a that phosphorylation of the and of phosphorylation on with the a that Aurora-A phosphorylation of p53 and S. S. Nat. Genet. data indicate that the phosphorylation of can have or a in on the phosphorylation site of p53 been identified the Our indicate that Aurora-A as as Ser-315, the Aurora-A abrogates p53 DNA binding and transactivation activity by phosphorylation of Ser-215 not we have that Ser-215 is a phosphorylation site of Aurora-A Ser-215 in and in to of p53 DNA binding and transactivation activity. As a result, the p53 target genes and and p53 tumor suppressor activity is inhibited by p53-S215A retained p53 function phosphomimic p53-S215D lost its tumor suppressor activity. of been in R. B. R. B. R. data indicate that phosphorylation of p53 at Ser-215 a mechanism for cell cycle cell and induced by to phosphorylation of Ser-215 in by p53 and the Ser-215 is phosphorylated by other serine/threonine protein kinases. Aurora is a subfamily of serine/threonine protein kinases. In vertebrates, they comprise Aurora-A, -B, and -C, with one or more highly conserved orthologues being found in the yeasts, flies, worms, and other invertebrates. Saccharomyces cerevisiae have a Aurora W. Biol. The and one in of the Aurora-A and R. Curr. Opin. Genet. The of Aurora-A and have been found in R. that Aurora a in progression of the cell to cell cycle such as and and and R. Curr. Opin. Genet. Aurora-A the that the is in been that of the Aurora-A in as as of and S. of Aurora-A in as as and S. have that Aurora-A important in cell and S. S. However, the molecular mechanism that Aurora-A p53 by phosphorylation of in S. S. Nat. Genet. we that DNA binding and transactivation activity of p53 was by Aurora-A p53 at Ser-215 in and in The of p53 DNA binding and transactivation activity by Aurora-A on phosphorylation of Ser-215 not Aurora-A phosphorylation of Ser-215 of p53 is with cell cycle cell and data indicate that p53 is a of Aurora-A and that Aurora-A its function phosphorylation of Ser-215 of In Ser-215 as a phosphorylation site of p53 that by other serine/threonine and p53 protein was were of p53 and Aurora-A were by of the of p53 and Aurora-A and of p53 were with a as a and in and were as Biol. In kinase was with in a or p53 as The was at for and in of p53 was by and were in and with or and in the to and a were and and were to the activity was by the activity to the activity. was and the and of p53, p53 was and the by in a and for and with a of the was to by with for at The was with of or for at by at in the of a of the and were as Biol. the the were by on a of The was for at in for the and for at in for the were with were by with in of p53 were by for at in The was by in The were by for at and the with The were to as to the phosphorylation sites Biol. were to and to of the the was with to and the and the was by was as S. Z. Biol. was with the p53 DNA binding site at for for p53 binding activity was in the of a of the or and of was by the of a for p53 The were by a and cell cycle were in in and with was by were with and a to the The of apoptosis was by was as were with p53 and Aurora-A or The was a and p53 protein was were of p53 and Aurora-A were by of the of p53 and Aurora-A and of p53 were with a as a and in and were as Biol. In kinase was with in a or p53 as The was at for and in of p53 was by and were in and with or and in the to and a were and and were to the activity was by the activity to the activity. was and the and of p53, p53 was and the by in a and for and with a of the was to by with for at The was with of or for at by at in the of a of the and were as Biol. the the were by on a of The was for at in for the and for at in for the were with were by with in of p53 were by for at in The was by in The were by for at and the with The were to as to the phosphorylation sites Biol. were to and to of the the was with to and the and the was by was as S. Z. Biol. was with the p53 DNA binding site at for for p53 binding activity was in the of a of the or and of was by the of a for p53 The were by a Cell and cell cycle were in in and with was by were with and a to the The of apoptosis was by was as were with p53 and Aurora-A or The was a p53 at Aurora-A with tumor suppressor p53 Aurora-A and p53 to and the of Aurora-A and of p53 function in of and R. S. S. Nat. Genet. Aurora-A with p53 that Aurora-A phosphorylated p53 in and in that Aurora-A phosphorylated p53 on serine p53 phosphorylated by Aurora-A, p53 was with and the were to by of the that the activity of was the cycle of the in the the not a the of p53, the that serine or at in its is phosphorylated serine indicate that Ser-215 is a phosphorylation site of p53 for Aurora-A result, p53 was with and and that the was Ser-215 and the other was and the of Ser-215 to Aurora-A phosphorylation of p53 in and in and Ser-215 of p53 is a major by p53 at of Aurora-A with cell were with and with and Aurora-A phosphorylation of p53 in and in were with of the were with The were to in kinase p53 as a The is in were with with or with in for and with The were by to to the and by of Aurora-A was with Aurora-A phosphorylation of p53 on serine The phosphorylated p53 protein by Aurora-A was in kinase and by as and p53 was with or and the were to of for cycle is in the been to at the R. in and the p53 that a serine in cycle of The a was and with that the that either or Aurora-A phosphorylation of p53 and the and in Aurora-A kinase and in were as p53-S215A as a p53 DNA and Ser-215 of p53, we Aurora-A DNA binding and transactivation activity of p53 that p53 DNA binding activity was inhibited by of Aurora-A in and of Aurora-A by the DNA binding activity The effect of Aurora-A on p53 transactivation activity was with p21Cip/WAF1 and in Aurora-A inhibited and activity in a the of Aurora-A by the activity of and were by of p53 and Aurora-A in protein of and were by of Aurora-A and by of Aurora-A data indicate that Aurora-A p53 and its DNA binding and transactivation of p53 and DNA on of Ser-215 phosphorylation of Ser-215 is for Aurora-A of p53 DNA binding and transactivation activity. Aurora-A phosphomimic p53-S215D and p53-S215A were by Ser-215 to and respectively. were in and that p53-S215A induced the and activity of and PTEN, to of However, of p53, were not inhibited by of phosphomimic p53-S215D lost the to induce and and activity. In p53-S215A retained DNA binding was not inhibited by Aurora-A, p53-S215D to to DNA we that Aurora-A of p53 on phosphorylation of that Aurora-A p53 at Ser-315, was in and to of p53 S. S. Nat. Genet. However, the its on p53 DNA binding and transactivation activity we phosphomimic as as and and that phosphomimic and retained p53 DNA binding and transactivation activity and were by Aurora-A and However, and to DNA binding data indicate that Aurora-A p53 phosphorylation of Ser-215 not of p53 on of p53 is a tumor suppressor and a of cell and cell cycle we the of Aurora-A phosphorylation of Ser-215 on p53 function. and have that to apoptosis and that p53 the response of the cell to cisplatin Biol. we the effect of Aurora-A phosphorylation of p53 on cell in The were with of p53, with and with and that of p53-S215A or p53 cisplatin and induced apoptosis at However, of Aurora-A inhibited the apoptosis induced by p53 not of apoptosis was in the and that were by In p53-S215D lost the to to cell p53, and not p53-S215D inhibited in and the cell Aurora-A and effect on p53-S215A In that the cell cycle at in p53, and were by Aurora-A and of Aurora-A, to the cell cycle arrest in with arrest was not in p53-S215D in response to is that of p53-S215D on cell and cell cycle progression as with with and data indicate that phosphorylation of Ser-215 not of p53 by Aurora-A a effect on p53 of p53 function phosphorylation of were with of p53 and with or of were with cisplatin for was by in and were with the of was was in and cell cycle with p53, Aurora-A, and were with at Cell cycle was in of p53 function phosphorylation of were with of p53 and with or of were with cisplatin for was by in and were with the of was was in and cell cycle with p53, Aurora-A, and were with at Cell cycle was in Aurora-A p53 at Aurora-A with tumor suppressor p53 Aurora-A and p53 to and the of Aurora-A and of p53 function in of and R. S. S. Nat. Genet. Aurora-A with p53 that Aurora-A phosphorylated p53 in and in that Aurora-A phosphorylated p53 on serine p53 phosphorylated by Aurora-A, p53 was with and the were to by of the that the activity of was the cycle of the in the the not a the of p53, the that serine or at in its is phosphorylated serine indicate that Ser-215 is a phosphorylation site of p53 for Aurora-A result, p53 was with and and that the was Ser-215 and the other was and the of Ser-215 to Aurora-A phosphorylation of p53 in and in and Ser-215 of p53 is a major by Aurora-A p53 DNA and Ser-215 of p53, we Aurora-A DNA binding and transactivation activity of p53 that p53 DNA binding activity was inhibited by of Aurora-A in and of Aurora-A by the DNA binding activity The effect of Aurora-A on p53 transactivation activity was with p21Cip/WAF1 and in Aurora-A inhibited and activity in a the of Aurora-A by the activity of and were by of p53 and Aurora-A in protein of and were by of Aurora-A and by of Aurora-A data indicate that Aurora-A p53 and its DNA binding and transactivation activity. Aurora-A of p53 and DNA on of Ser-215 phosphorylation of Ser-215 is for Aurora-A of p53 DNA binding and transactivation activity. Aurora-A phosphomimic p53-S215D and p53-S215A were by Ser-215 to and respectively. were in and that p53-S215A induced the and activity of and PTEN, to of However, of p53, were not inhibited by of phosphomimic p53-S215D lost the to induce and and activity. In p53-S215A retained DNA binding was not inhibited by Aurora-A, p53-S215D to to DNA we that Aurora-A of p53 on phosphorylation of that Aurora-A p53 at Ser-315, was in and to of p53 S. S. Nat. Genet. However, the its on p53 DNA binding and transactivation activity we phosphomimic as as and and that phosphomimic and retained p53 DNA binding and transactivation activity and were by Aurora-A and However, and to DNA binding data indicate that Aurora-A p53 phosphorylation of Ser-215 not Aurora-A of p53 on of p53 is a tumor suppressor and a of cell and cell cycle we the of Aurora-A phosphorylation of Ser-215 on p53 function. and have that to apoptosis and that p53 the response of the cell to cisplatin Biol. we the effect of Aurora-A phosphorylation of p53 on cell in The were with of p53, with and with and that of p53-S215A or p53 cisplatin and induced apoptosis at However, of Aurora-A inhibited the apoptosis induced by p53 not of apoptosis was in the and that were by In p53-S215D lost the to to cell p53, and not p53-S215D inhibited in and the cell Aurora-A and effect on p53-S215A In that the cell cycle at in p53, and were by Aurora-A and of Aurora-A, to the cell cycle arrest in with arrest was not in p53-S215D in response to is that of p53-S215D on cell and cell cycle progression as with with and data indicate that phosphorylation of Ser-215 not of p53 by Aurora-A a effect on p53 function. have that Aurora-A proteins that important for proteins a in of the to mitotic C. S. Biol. for its in of a protein for and of R. S. C. Cell Biol. a protein in and and stability S. Biol. and is to R. Cell Biol. In Ser-215 of p53 been identified as a phosphorylation site of Aurora-A phosphorylation of p53 p53 DNA binding and transactivation activity. As a result, p53 target such as and PTEN, were by function and cell cycle arrest in response to DNA were by important for they provide a of the Aurora-A function in cell cell and a Aurora-A and the DNA been is the of a phosphorylation site p53 is of the that its activity as a transactivation the conserved a and a been that phosphorylation of p53 in of the of to p53 in the and of the of p53, to its phosphorylation of p53 the at or can the or protein B. R. S. S. Biol. phosphorylation of p53 protein at sites DNA or can the of C. the of p53 in the is a posttranslational important in the activity of p53 in of the DNA binding function of p53 in and in the of the of not of been that is a of and C. S. C. of p53 at site the DNA binding activity of p53 in C. B. a that phosphorylation of the and of phosphorylation on with the a that Aurora-A phosphorylation of p53 and S. S. Nat. Genet. data indicate that the phosphorylation of can have or a in on the phosphorylation site of p53 been identified the Our indicate that Aurora-A as as Ser-315, the Aurora-A abrogates p53 DNA binding and transactivation activity by phosphorylation of Ser-215 not we have that Ser-215 is a phosphorylation site of Aurora-A Ser-215 in and in to of p53 DNA binding and transactivation activity. As a result, the p53 target genes and and p53 tumor suppressor activity is inhibited by p53-S215A retained p53 function phosphomimic p53-S215D lost its tumor suppressor activity. of been in R. B. R. B. R. data indicate that phosphorylation of p53 at Ser-215 a mechanism for cell cycle cell and induced by to phosphorylation of Ser-215 in by p53 and the Ser-215 is phosphorylated by other serine/threonine protein kinases. have that Aurora-A proteins that important for proteins a in of the to mitotic C. S. Biol. for its in of a protein for and of R. S. C. Cell Biol. a protein in and and stability S. Biol. and is to R. Cell Biol. In Ser-215 of p53 been identified as a phosphorylation site of Aurora-A phosphorylation of p53 p53 DNA binding and transactivation activity. As a result, p53 target such as and PTEN, were by function and cell cycle arrest in response to DNA were by important for they provide a of the Aurora-A function in cell cell and a Aurora-A and the DNA been is the of a phosphorylation site p53 p53 is of the that its activity as a transactivation the conserved a and a been that phosphorylation of p53 in of the of to p53 in the and of the of p53, to its phosphorylation of p53 the at or can the or protein B. R. S. S. Biol. phosphorylation of p53 protein at sites DNA or can the of C. the of p53 in the is a posttranslational important in the activity of p53 in of the DNA binding function of p53 in and in the of the of not of been that is a of and C. S. C. of p53 at site the DNA binding activity of p53 in C. B. a that phosphorylation of the and of phosphorylation on with the a that Aurora-A phosphorylation of p53 and S. S. Nat. Genet. data indicate that the phosphorylation of can have or a in on the phosphorylation site of p53 been identified the Our indicate that Aurora-A as as Ser-315, the Aurora-A abrogates p53 DNA binding and transactivation activity by phosphorylation of Ser-215 not In we have that Ser-215 is a phosphorylation site of Aurora-A Ser-215 in and in to of p53 DNA binding and transactivation activity. As a result, the p53 target genes and and p53 tumor suppressor activity is inhibited by p53-S215A retained p53 function phosphomimic p53-S215D lost its tumor suppressor activity. of been in R. B. R. B. R. data indicate that phosphorylation of p53 at Ser-215 a mechanism for cell cycle cell and induced by to phosphorylation of Ser-215 in by p53 and the Ser-215 is phosphorylated by other serine/threonine protein kinases. to the DNA and at