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Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARSI replicator contains multiple functional DNA elements: an essential A element and three important B elements-Bi, B2, and B3. Functionally similar A, Bi, and B2 elements are also present in the ARS307 replicator. Eukaryotic cells duplicate their genetic information for the next generation during the S phase of the cell division cycle. In general, two steps are required for a cell to duplicate its genome. First, a multiprotein complex is assembled at specific sites within the chromosome where DNA replication starts (the replication origins). Second, the enzymatic machinery that replicates the DNA is activated and replication forks move bidirectionally to synthesize the new DNA chains (1). The replication process is highly regulated at the initiation step. Studies in bacteria and with viruses that infect mammalian cells support the replicon model (2), which posits that DNA replication initiates at a specific site (origin) and this initiation is controlled by a cis-acting DNA sequence called the replicator (1-3). A replicator-specific DNA binding protein called the initiator facilitates the assembly of the enzymatic machinery at the replication origin to allow initiation of DNA replication to proceed.The nature of replicators and the proteins that are involved in the initiation of DNA replication are the keys to understanding the mechanism and regulation of initiation of eukaryotic DNA replication. Autonomous replicating sequences (ARSs) were identified in the yeast Saccharomyces cerevisiae by their ability to promote the maintenance of extrachromosomal DNA (4-7). Subsequently, both replicators and origins of DNA replication in the chromosome were found to colocalize with a subset of these ARS elements (7). Fine structure analysis of the ARS1 and ARS307 replicators demonstrated that they contain multiple short functional elements (8)(9)(10) (7,8). The B3 element is found in some but not all of the ARSs and it functions in an orientation-and position-independent manner to enhance origin utilization (12, 13).A multisubunit origin recognition complex (ORC) was identified as a candidate initiator protein using the DNase I footprint method and was shown to bind toARSJ and all other ARS sequences in an ATP-dependent manner (14,15). ORC contains six subunits of 120, 72, 62, 56, 53, and 50 kDa. Genes encoding all six subunits have been cloned and found to be essential for cell viability (S. P. Bell, R. Kobayashi, and B.S., unpublished data; see refs. 15-18), Further genetic analyses suggest that ORC plays an important role in DNA replication (reviewed in ref. 7). The ORC DNase I protection pattern at ARS sequences extends over a 50-bp region that includes the A and Bi elements (9,14). In addition, several DNase I-hypersensitive sites are induced in the DNA. These hypersensitive sites are found at 10-bp intervals, suggesting that the DNA may wrap aro...

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.