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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
Nader Shakibai, Vijay Kumar and Shlomo Eisenberg
Proceedings of the National Academy of Sciences of the United States of America
Vol. 93, No. 21 (Oct. 15, 1996), pp. 11569-11574
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/40475
Page Count: 6
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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.
Proceedings of the National Academy of Sciences of the United States of America © 1996 National Academy of Sciences