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 stm1⌬ yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acidbinding 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.It has long been recognized that, under the proper conditions, certain DNA sequences preferentially adopt a structure composed of three nucleic acid strands (1). Triple helical or triplex DNA is a thermodynamically favored structure characterized by a third pyrimidine-rich (Py triplex) 1 or purine-rich (Pu triplex) DNA strand located within the major groove of a homopurine/homopyrimidine stretch of duplex DNA (reviewed in Ref. 2). Both intermolecular triplexes, where the third stand originates from a separate DNA molecule, and intramolecular triplexes (H-DNA), where the third stand originates from a proximal site on the same DNA molecule as its duplex acceptor, have been described. In intermolecular and intramolecular triplexes, stable interaction of the third strand is achieved through either specific Hoogsteen (Py triplex) or reverse Hoogsteen (Pu triplex) hydrogen bonding to the homopurine strand of the duplex, with the third strand adopting either a parallel (Py triplex) or antiparallel (Pu triplex) orientation relative to the homopurine acceptor. Base triplets in the pyrimidine motif include T*AT and C ϩ