The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.
We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.
Shakibai
N
N
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Kumar
V
V
Eisenberg
S
S
D000818:Animals; D034961:Antigens, Nuclear; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D000072200:Ku Autoantigen; D008322:Mammals; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D034961:Antigens, Nuclear; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; C520769:YKU70 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen