Modulation of the Active Complex Assembly and Turnover Rate by Protein-DNA Interactions in Cre-LoxP Recombination
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- 作者：Shelley S. Martin ; Victor C. Chu ; and Enoch Baldwin
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：June 10, 2003
- 年：2003
- 卷：42
- 期：22
- 页码：6814 - 6826
- 全文大小：674K
- 年卷期：v.42,no.22(June 10, 2003)
- ISSN：1520-4995
文摘
Cre promotes recombination at the 34 bp LoxP sequence. Substitution of a critical C-G basepair in LoxP with an A-T base pair, to give LoxAT, reduced Cre binding in vitro and abolishedrecombination in vivo [Hartung, M., and Kisters-Woike, B. (1998) J. Biol. Chem. 273, 22884-22891].We demonstrated that LoxAT can be recombined in vitro. However, Cre discriminates against this substrateboth before and after DNA binding. The preference for LoxP over LoxAT is the result of reduced bindingand a slower turnover rate, amplified by changes in cooperativity of complex assembly. With LoxAT,similar levels of substrate turnover required 2-2.5-fold higher protein-DNA concentrations comparedto LoxP, but the sigmoidal behavior of the concentration dependence was more pronounced. Further, theCre-LoxAT complexes reacted 4-5-fold more slowly. In the 2.3 Å resolution Cre-LoxAT complexstructure, the major groove Arg259-guanine interaction was disrupted, explaining the reduced binding.Overall structural shifts and mobility changes indicate more favorable interactions between subunits,providing a hypothesis for the reduced turnover rate. Concomitant with the displacement of Arg259 fromthe DNA, adjacent charged residues Glu262 and Glu266 shifted to form salt bridges with the Arg259guanidinium moiety. Substitution of Glu262 and Glu266 with glutamine increased Cre complex assemblyefficiency and reaction rates with both LoxAT and LoxP, but diminished Cre's ability to distinguishthem. The increased rate of this variant suggests that DNA substrate binding and turnover are coupled.The improved efficiency, made at some expense of sequence discrimination, may be useful for enhancingrecombination in vivo.