Title

Purification and characterization of proteins required for the assembly of a sequence-specific complex at a yeast origin of replication

Date of Completion

January 1999

Keywords

Biology, Molecular|Biology, Cell|Biology, Microbiology

Degree

Ph.D.

Abstract

Previous work from our laboratory has demonstrated 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 (Proc. Natl. Acad. Sci., USA. 1992. 89(23): 11156–60.). One of these three activities is the known replication enhancer protein ABF1. In this work we show the purification to homogeneity of the activity referred to as OBF2, and the partial purification of the other unknown factor, CBF. The purified OBF2 is a heterodimer composed of two polypeptides with apparent molecular mass values of 65 and 80 kDa. Purified OBF2 not only binds DNA but also supports the formation of a CBF-containing 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 double-stranded DNA termini. The requirement of OBF2 for the formation of the sequence-specific protein complex at ARS121 is not satisfied by the binding of OBF2 to the DNA termini only. ^ Amino 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 previously 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. ^ Although the CBF activity has not yet been purified to homogeneity, it shares some of the physical and biochemical properties of the origin recognition complex (ORC), a six subunit protein known to interact with essential sequences at a variety of yeast origins (Nature. 1992. 357(6374): 128–134.). The potential significance of the multiprotein complex that we have formed in vitro is discussed in relation to the known activities of the Ku-like proteins as well as the function of protein-DNA complexes at chromosomal origins of replication. ^