title: Analysis of nucleotide sequence-dependent DNA binding of poly(ADP-ribose) polymerase in a purified system.
abstract: The enzymatic transfer of ADP-ribose from NAD to histone H(1) [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 oligonucleotides of dsDNA can serve as coenzymic DNA for PARP-1 trans- or auto-modification activity. Results of DNA-protein binding (EMSA) experiments reported here demonstrate that short DNA oligonucleotides containing 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 a contractile protein gene encoding cardiac troponin T (cTnT). Results of experiments reported here demonstrate that mutation of this motif also abolishes the differentiation-dependent activation of the transfected cTnT promoter in myoblasts cultured in vitro, indicating that nucleotide sequence-dependent binding of PARP-1 to promoter DNA of the cTnT gene is also necessary for differentiation-dependent activation. Thus, PARP-1 has two types of dsDNA binding activity: (1) nucleotide sequence-dependent binding, analyzed here with EMSA experiments, and (2) coenzymic binding, measured catalytically, which does not depend on the nucleotide sequence of the dsDNA. We hypothesize that the well-known association of PARP-1 with chromatin can be attributed to its stable binding to chromosomal dsDNA, some portion of which is likely to be nucleotide sequence-dependent binding. According to this hypothesis, the distribution of this protein-modifying enzyme in chromatin may be targeted to specific genomic loci and vary according to cell type and developmental stage.
authors.affiliation: Department of Anatomy and The Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94143, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.forename: William E
authors.initials: WE
authors.lastname: Tidyman
authors.forename: Kim-Uyen T
authors.initials: KU
authors.lastname: Le
authors.forename: Eva
authors.initials: E
authors.lastname: Kirsten
authors.forename: Ernest
authors.initials: E
authors.lastname: Kun
authors.forename: Charles P
authors.initials: CP
authors.lastname: Ordahl
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000502:physiology; D001667:Binding, Competitive; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002645:Chickens; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011784:Quail; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000737:chemistry; D014162:Transfection; D020107:Troponin T / Q000737:chemistry / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D020107:Troponin T; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases