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首页> 外文期刊>Molecular and Cellular Biology >Discrimination of DNA binding sites by mutant p53 proteins.
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Discrimination of DNA binding sites by mutant p53 proteins.

机译：通过突变的p53蛋白区分DNA结合位点。

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Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.
机译：通过分子遗传学方法已经确定了p53识别DNA的关键决定因素。将包含氨基酸71至330的野生型人p53片段（p53（71-330））用于体外DNA结合测定，将全长人p53用于酿酒酵母的反式激活测定。首先，我们通过使用已知共有DNA位点的系统改变形式，定义了野生型p53片段的DNA结合特异性。这种改进表明p53与PuGPuCA.TGPyCPy的两个重复序列高亲和力结合，这是对先前定义的共有半位点PuPuPuC（A / T）。（T / A）GPyPyPy的进一步改进。通过使用全长人p53和系统改变的DNA位点，通过酵母的反式激活试验进一步证实了这些结果。五聚体AGGGA的二聚体可有效地头对头或尾对尾结合，但仅通过头对头二聚体促进反式激活。为了确定p53与DNA结合的特异性起源，我们鉴定了导致DNA结合特异性改变的突变。在p53的DNA结合域内的多个位置进行了单氨基酸取代，并用DNA位点变异体测试了这套p53点突变体的DNA结合。 DNA结合分析表明，突变体Lys-120突变为Asn，Cys-277突变为Gln或Arg，以及Arg-283突变为Gln结合到五聚体（PuGPuCA）中位置2、3和1处具有非经典碱基对的位点，分别。因此，我们将这些残基暗示为氨基酸-碱基对接触。有趣的是，与Gln结合的突变型Cys-277以两个和四个单体结合一个共有位点，而不是野生型p53片段，后者始终以四个单体结合该位点。

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