The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature‐sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi‐ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5-DNA kinase and 3-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5-OH termini was only ϳ5-fold tighter than that to identical DNA molecules with 5-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5-OH termini and those with 5-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.Scission of the DNA sugar-phosphate backbone is a common form of damage that can be induced not only by a broad range of genotoxic agents, but also as an intermediate product in several DNA repair pathways. The term "strand break" covers an array of diverse chemical structures. Aside from single-and doublestrand breaks, there are many chemically distinct end groups found at strand break termini. Repair of these strand interruptions is usually mediated by DNA polymerases and ligases. All DNA polymerases and ligases characterized to date are highly selective for the type of DNA ends that can be utilized. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by several endonucleases, as well as those induced by ionizing radiation, frequently bear 5Ј-hydroxyl and/or 3Ј-phosphate groups (1-5) and must therefore be processed before they can be acted upon by DNA ligases or polymerases.Mammalian polynucleotide kinase (PNK) 3 /phosphatase is a bifunctional enzyme that can phosphorylate 5Ј-OH termini and dephosphorylate 3Ј-phosphate termini of DNA (6, 7). It is a DNA repair enzyme involved in the processing of strand break termini to a form suitable for other proteins to complete the replacement of missing nucleotides and strand rejoining (8 -11). PNK is implicated in the repair of both single-strand breaks (SSBs) and double-strand breaks. PNK stimulates SSB repair in both in vitro reconstitution experiments (5,10,12,13) and in vivo studies (14). Further evidence has indicated that, as a result of its required involvement in the repair of specific types of SSBs, PNK participates in the base excision repair pathway following the formation of strand breaks induced by DNA glycosylases such as NEIL1 and NEIL2 (5) and in the repair of topoisomerase-1 "dead-end" complexes (15,16). PNK i...

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2k or H-2d haplotypes, and Ab-blocking experiments indicated that H-2Dk and Dd are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2k class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.

The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2Dd and Dk class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes Dd, and not Dk. The specificity for Dk as well as Dd of the wild-type Ly-49GBALB/c allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49GBALB/c allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49GB6. Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.