Analysis of Nucleotide Sequence-Dependent DNA Binding of Poly(ADP-ribose) Polymerase in a Purified System

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• 作者：Kai Huang ; William E. Tidyman ; Kim-Uyen T. Le ; Eva Kirsten ; Ernest Kun ; and Charles P. Ordahl
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：January 13, 2004
• 年：2004
• 卷：43
• 期：1
• 页码：217 - 223
• 全文大小：166K
• 年卷期：v.43,no.1(January 13, 2004)
• ISSN：1520-4995

文摘

The enzymatic transfer of ADP-ribose from NAD to histone H₁ [defined as trans(oligo-ADP-ribosylation)] or to PARP-1 [defined as auto(poly-ADP-ribosylation)] requires binding of coenzymic DNA.The preceding paper [Kun, E., et al. (2004) Biochemistry 43, 210-216] shows that oligonucleotidesof dsDNA can serve as coenzymic DNA for PARP-1 trans- or auto-modification activity. Results ofDNA-protein binding (EMSA) experiments reported here demonstrate that short DNA oligonucleotidescontaining the 5'-TGTTG-3' nucleotide sequence motif preferentially bind to cloned PARP-1 in vitro.The same nucleotide sequence motif is responsible for striated myocyte-selective transcription of acontractile protein gene encoding cardiac troponin T (cTnT). Results of experiments reported heredemonstrate that mutation of this motif also abolishes the differentiation-dependent activation of thetransfected cTnT promoter in myoblasts cultured in vitro, indicating that nucleotide sequence-dependentbinding of PARP-1 to promoter DNA of the cTnT gene is also necessary for differentiation-dependentactivation. Thus, PARP-1 has two types of dsDNA binding activity: (1) nucleotide sequence-dependentbinding, analyzed here with EMSA experiments, and (2) coenzymic binding, measured catalytically, whichdoes not depend on the nucleotide sequence of the dsDNA. We hypothesize that the well-known associationof PARP-1 with chromatin can be attributed to its stable binding to chromosomal dsDNA, some portionof which is likely to be nucleotide sequence-dependent binding. According to this hypothesis, the distributionof this protein-modifying enzyme in chromatin may be targeted to specific genomic loci and vary accordingto cell type and developmental stage.