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Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.

Cai BH, Chen JY, Lu MH, Chang LT, Lin HC, Chang YM, Chao CF - Nucleic Acids Res. (2009)

Bottom Line: The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence.A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG.Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

View Article: PubMed Central - PubMed

Affiliation: National Defense Medical Center, Institute of Life Sciences, Taipei, Taiwan, ROC.

ABSTRACT
The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

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The p53 consensus sequence. The purine (R) and pyrimidine (Y) bases comprise the flanking sequence. C(A/T)(A/T)G is the core sequence, and the (A/T)(A/T) within the core sequence is an A/T gap. The whole site is 20 bases with a ten-base direct repeat as a 2× half site, and each 10 bases are a 1× half site. Each five bases are quarter sites. A whole site with a core sequence C to G mutation within the first quarter site is a 1.5× half site, with a core sequence G to C mutation within the fourth quarter site is another 1.5× half site.
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Figure 1: The p53 consensus sequence. The purine (R) and pyrimidine (Y) bases comprise the flanking sequence. C(A/T)(A/T)G is the core sequence, and the (A/T)(A/T) within the core sequence is an A/T gap. The whole site is 20 bases with a ten-base direct repeat as a 2× half site, and each 10 bases are a 1× half site. Each five bases are quarter sites. A whole site with a core sequence C to G mutation within the first quarter site is a 1.5× half site, with a core sequence G to C mutation within the fourth quarter site is another 1.5× half site.

Mentions: p53 is a key regulator of the cell cycle. After DNA damage, p53 can be activated to block cell-cycle progression and mediate multiple check points. The p53 target genes, p21 and 14-3-3σ, can be induced by activated p53 to arrest the G1-to-S phase and G2-to-M phase of the cell cycle, respectively (1,2). The p53 consensus sequence is 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases (3). C(A/T)(A/T)G is the core sequence, and the purine (pu) and pyrimidine (py) bases comprise the flanking sequence (Figure 1). The whole p53-binding sequence is the 10-base direct repeated sequence. Only 10 and 5 bases comprise the half site and quarter site of the p53-binding site, respectively. The p53-binding sequence with a half site plus a quarter site is equal to 1.5-fold of the p53 half site (4). A half site of the p53-binding site possesses p53-binding affinity (4); however, it can only be activated by p53 in a sequence-specific manner or with other required cofactors (5–7). About 1.5-fold of the p53 half site can be activated by p53, but its activity is much lower than for the whole p53-binding site (5,7). Both p21 and 14-3-3σ have two p53 consensus sequences on their promoter (8). The p21 promoter at 5′ site one (GAACATGTCCcAACATGTTg) and 3′ site two (GAagAAGaCTGGGCATGTCT) can be activated by p53. The 14-3-3σ promoter 3′ site two (GtAGCAttAGCCCAGACATGTCC) can be activated by p53 but not that at 5′ site one (AGGCATGTgCcAcCATGCCC) (9). However, the p21 and 14-3-3σ promoter p53-binding sites presented some mismatched bases (the lowercase letters note the mismatch bases). There are three mismatch bases in the flanking sequence of the p53-binding site in 14-3-3σ site one, but only one mismatch base in 14-3-3σ site two. There are two bases of the A/T gap in the core sequence of the p53-binding site in p21 site one and 14-3-3σ site one, but there are four bases in 14-3-3σ site two. Although the entire p53 consensus sequence comprises the whole p53-binding site, a 1.5-fold half site of the p53-binding site could be activated by p53, similar to p21 site two (7). In order to identify whether the p53-regulated site on the 14-3-3σ promoter is a whole site or a 1.5-fold half site, different mutant core sequences were cloned and tested for their functional activities. Interestingly, the p53-binding site on the 14-3-3σ promoter contained four bases of the A/T gap CATTAG core sequence. It differed from the p53 consensus by two bases of A/T gap CATG core sequence. To clarify how many bases of the A/T gap in the p53 core sequence are tolerant for p53-activating genes, basic promoter vectors with different A/T gap p53 response elements were constructed. The promoter activities and -binding affinities were measured in our experiments.Figure 1.


Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.

Cai BH, Chen JY, Lu MH, Chang LT, Lin HC, Chang YM, Chao CF - Nucleic Acids Res. (2009)

The p53 consensus sequence. The purine (R) and pyrimidine (Y) bases comprise the flanking sequence. C(A/T)(A/T)G is the core sequence, and the (A/T)(A/T) within the core sequence is an A/T gap. The whole site is 20 bases with a ten-base direct repeat as a 2× half site, and each 10 bases are a 1× half site. Each five bases are quarter sites. A whole site with a core sequence C to G mutation within the first quarter site is a 1.5× half site, with a core sequence G to C mutation within the fourth quarter site is another 1.5× half site.
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Related In: Results  -  Collection

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Figure 1: The p53 consensus sequence. The purine (R) and pyrimidine (Y) bases comprise the flanking sequence. C(A/T)(A/T)G is the core sequence, and the (A/T)(A/T) within the core sequence is an A/T gap. The whole site is 20 bases with a ten-base direct repeat as a 2× half site, and each 10 bases are a 1× half site. Each five bases are quarter sites. A whole site with a core sequence C to G mutation within the first quarter site is a 1.5× half site, with a core sequence G to C mutation within the fourth quarter site is another 1.5× half site.
Mentions: p53 is a key regulator of the cell cycle. After DNA damage, p53 can be activated to block cell-cycle progression and mediate multiple check points. The p53 target genes, p21 and 14-3-3σ, can be induced by activated p53 to arrest the G1-to-S phase and G2-to-M phase of the cell cycle, respectively (1,2). The p53 consensus sequence is 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases (3). C(A/T)(A/T)G is the core sequence, and the purine (pu) and pyrimidine (py) bases comprise the flanking sequence (Figure 1). The whole p53-binding sequence is the 10-base direct repeated sequence. Only 10 and 5 bases comprise the half site and quarter site of the p53-binding site, respectively. The p53-binding sequence with a half site plus a quarter site is equal to 1.5-fold of the p53 half site (4). A half site of the p53-binding site possesses p53-binding affinity (4); however, it can only be activated by p53 in a sequence-specific manner or with other required cofactors (5–7). About 1.5-fold of the p53 half site can be activated by p53, but its activity is much lower than for the whole p53-binding site (5,7). Both p21 and 14-3-3σ have two p53 consensus sequences on their promoter (8). The p21 promoter at 5′ site one (GAACATGTCCcAACATGTTg) and 3′ site two (GAagAAGaCTGGGCATGTCT) can be activated by p53. The 14-3-3σ promoter 3′ site two (GtAGCAttAGCCCAGACATGTCC) can be activated by p53 but not that at 5′ site one (AGGCATGTgCcAcCATGCCC) (9). However, the p21 and 14-3-3σ promoter p53-binding sites presented some mismatched bases (the lowercase letters note the mismatch bases). There are three mismatch bases in the flanking sequence of the p53-binding site in 14-3-3σ site one, but only one mismatch base in 14-3-3σ site two. There are two bases of the A/T gap in the core sequence of the p53-binding site in p21 site one and 14-3-3σ site one, but there are four bases in 14-3-3σ site two. Although the entire p53 consensus sequence comprises the whole p53-binding site, a 1.5-fold half site of the p53-binding site could be activated by p53, similar to p21 site two (7). In order to identify whether the p53-regulated site on the 14-3-3σ promoter is a whole site or a 1.5-fold half site, different mutant core sequences were cloned and tested for their functional activities. Interestingly, the p53-binding site on the 14-3-3σ promoter contained four bases of the A/T gap CATTAG core sequence. It differed from the p53 consensus by two bases of A/T gap CATG core sequence. To clarify how many bases of the A/T gap in the p53 core sequence are tolerant for p53-activating genes, basic promoter vectors with different A/T gap p53 response elements were constructed. The promoter activities and -binding affinities were measured in our experiments.Figure 1.

Bottom Line: The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence.A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG.Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

View Article: PubMed Central - PubMed

Affiliation: National Defense Medical Center, Institute of Life Sciences, Taipei, Taiwan, ROC.

ABSTRACT
The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

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