The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitinconjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.

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The SCAN box or leucine-rich (LeR) domain is a conserved motif found within a subfamily of C 2 H 2 zinc finger proteins. The function of a SCAN box is unknown, but it is predicted to form ␣-helices that may be involved in protein-protein interactions. Myeloid zinc finger gene-1B (MZF1B) is an alternatively spliced human cDNA isoform of the zinc finger transcription factor, MZF1. MZF1 and MZF1B contain 13 C 2 H 2 zinc finger motifs, but only MZF1B contains an amino-terminal SCAN box. A bone marrow cDNA library was screened for proteins interacting with the MZF1B SCAN box domain and RAZ1 (SCAN-related protein associated with MZF1B) was identified. RAZ1 is a novel cDNA that encodes a SCAN-related domain and arginine-rich region but no zinc finger motifs. Co-immunoprecipitation assays demonstrate that the SCAN box domain of MZF1B is necessary for association with RAZ1. By yeast twohybrid analysis, the carboxyl terminus of RAZ1 is sufficient for interaction with the MZF1B SCAN box. Furthermore, MZF1B and RAZ1 each self-associate in vitro via a SCAN box-dependent mechanism. These data provide evidence that the SCAN box is a protein interaction domain that mediates both hetero-and homoprotein associations.Zinc finger genes encode an abundant class of DNA-and RNA-binding proteins that represent an estimated 5% of the genes in the human genome. Many C 2 H 2 zinc finger genes have been demonstrated to function as transcriptional regulators and frequently, zinc finger genes are targeted for disruption in a variety of human diseases and cancers. The Krü ppel-like subclass of mammalian C 2 H 2 zinc finger proteins, first identified in the zinc finger transcription factor TFIIIA, share a conserved link between the last histidine of the preceding finger motif with the first cysteine of the next finger (H-C link) (1). Krü ppel-like proteins often contain conserved modular domains outside of their zinc finger motifs. These identified domains include the KRAB (Krü ppel-associated box) domains A and B, FAX (finger-associated box) domain of Xenopus, BTB/ POZ (broad complex, tramtrack, and bric-a-brac/poxvirus and zinc finger) or ZiN (zinc finger N-terminal) domain, and the SCAN box or leucine-rich domain.To date, the functions of the KRAB and BTB/POZ domains have been best characterized. The KRAB domain is a conserved stretch of 75 amino acids found in an estimated one-third of Krü ppel-like zinc finger proteins (2). The KRAB domain, further subdivided into domains A and B, functions as a potent transcriptional repressor (3-5) and is predicted to fold into two amphipathic helices (2). The KRAB domain from KOX1 interacts with human TIF1␤ (also named KAP-1, KRAB-associated protein-1) (6, 7) and appears to exert its transcriptional repression activity through this interaction (6, 7). In addition, the KRAB-A domain of Kid-1 interacts with KRIP-1 (KRAB-A interacting protein), which is likely to be the murine homologue of TIF1␤ and KAP-1 (8). The POZ domain defines a conserved region of approximately 120 amino acids and is found in 5-10%...

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