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首页> 外文期刊>Journal of Molecular Biology >Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre.
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Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre.

机译：Flp和Cre重组酶的嵌合体：测试Flp和Cre的切割方式。

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摘要

The Flp and Cre recombinases are members of the integrase family of tyrosine recombinases. Each protein consists of a 13 kDa NH(2)-terminal domain and a larger COOH-terminal domain that contains the active site of the enzyme. The COOH-terminal domain also contains the major determinants for the binding specificity of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attachment of a conserved nucleophilic tyrosine residue to the 3'-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding specificity and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH(2)-terminal domain of Flp to the COOH-terminal domain of Cre ("Fre") and the NH(2)-terminal domain of Cre to the COOH-terminal domain of Flp ("Clp"). These chimeras have novel binding specificities in that they bind strongly to hybrid sites containing elements from both the Flp and Cre DNA targets but poorly to the native target sites.In this study we have taken advantage of the unique binding specificities of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We find that the COOH-terminal domain of the recombinases determines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous findings that Cre could carry out trans cleavage. We discuss the variations in the experimental approaches in order to reconcile the different results. Copyright 2000 Academic Press.
机译：Flp和Cre重组酶是酪氨酸重组酶整合酶家族的成员。每个蛋白质由一个13 kDa NH（2）末端域和一个较大的COOH末端域组成，后者包含酶的活性位点。 COOH-末端结构域还包含重组酶与其同源DNA结合位点的结合特异性的主要决定因素。所有家族成员都通过在切割位点将保守的亲核酪氨酸残基连接到3'-磷酸基团来切割DNA。为了进一步了解Flp和Cre的结合特异性和切割方式的决定因素，我们制备了嵌合蛋白，其中我们将Flp的NH（2）末端结构域融合到Cre的COOH末端结构域（“ Fre”）和Cre的NH（2）末端结构域到Flp的COOH末端结构域（“ Clp”）。这些嵌合体具有新颖的结合特异性，因为它们与包含Flp和Cre DNA靶标的元素的杂合位点牢固结合，而与天然靶位点结合力很弱。在这项研究中，我们利用Fre和Clp独特的结合特异性来检测Cre，Flp，Fre和Clp的切割方式。我们发现重组酶的COOH-末端结构域决定了它们的切割方式。因此，Flp和Clp裂解为反式，而Cre和Fre裂解为顺式。这些结果与Flp的研究以及与Cre结合至其DNA靶位点的Cre的共晶体结构一致。他们不同意我们以前的发现，即Cre可以进行反式切割。我们讨论实验方法中的变化，以调和不同的结果。版权所有2000学术出版社。

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