Abstract
The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiae whole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K(d) of 61 pM) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Delta yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1, MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed.
Highlights
It has long been recognized that, under the proper conditions, certain DNA sequences preferentially adopt a structure composed of three nucleic acid strands [1]
Using electrophoretic mobility shift assays (EMSA), we have found evidence for Pu motif 3BPs in extracts from organisms ranging from bacteria to human
The C1 species was reduced to 64% and less than 3% of its normal amount when 100- and 200-fold molar excess unlabeled, noncovalent Pu triplex DNA was present in the binding reaction, respectively
Summary
Pyrimidine; Pu, purine; 3BP, triplex-binding protein; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; TFO, triplex-forming oligodeoxyribonucleotide; G4, G-quartet; FPLC, fast protein liquid chromatography; X, photo-cross-linked. Direct proof is lacking, long oligopurine tracts with triplex-forming potential are quite common in eukaryotic genomes, ranging from yeast to human [5] These tracts are distributed nonrandomly and are typically located near gene promoters, recombination hot spots, and matrix attachment regions [6, 7]. Monoclonal antibodies generated against triplex DNA were found to interact nonuniformly with metaphase chromosomes and interphase nuclei, preferentially staining centromeric regions [14, 15] Taken together, these data support the existence of triplex DNA at some point during the life cycle of a eukaryotic cell and suggest an important role for these structures in DNA-dependent biological processes. Using electrophoretic mobility shift assays (EMSA), we have found evidence for Pu motif 3BPs in extracts from organisms ranging from bacteria to human.2 These data suggest that 3BPs are present in all eukaryotes and that they play important cellular roles. We describe the purification and characterization of the major S. cerevisiae 3BP, y3BP1, and its identification as the product of the STM1 gene
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