Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination.

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae.

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.

Identification of a novel membrane transporter associated with intracellular membranes by phenotypic complementation in the yeast Saccharomyces cerevisiae.

A partial mouse cDNA was isolated by its ability to functionally complement a thymidine transport deficiency in plasma membranes of the yeast, Saccharomyces cerevisiae. The full-length cDNA encoded a previously unidentified 27-kDa protein (mouse transporter protein (MTP)) with four predicted transmembrane-spanning domains. MTP mRNA was detected in cells of several mammalian species, and its predicted protein sequence exhibited near identity (98%) with that of a human cDNA (HUMORF13). MTP and its homologs evidently reside in an intracellular membrane compartment because a protein (about 24 kDa) that was recognized by MTP-specific antibodies was observed in a subcellular fraction of rat hepatocytes enriched for Golgi membranes. Deletion of the hydrophilic C terminus of MTP, which encompassed two putative signal motifs for intracellular localization (Tyr-X-X-hydrophobic amino acid), allowed expression of recombinant protein (MTP deltaC) in plasma membranes of Xenopus laevis oocytes. MTP deltaC-expressing oocytes exhibited greater fragility than nonexpressing oocytes, and those that survived the experimental manipulations were capable of mediated uptake of thymidine, uridine, and adenosine. Thymidine uptake by MTP deltaC-expressing oocytes was inhibited by thymine and dTMP. MTP may function in the transport of nucleosides and/or nucleoside derivatives between the cytosol and the lumen of an intracellular membrane-bound compartment.

Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.

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)

Vancomycin, metronidazole, and tetracyclines.

Over the last 30 years, the impurities in the vancomycin product have been reduced, perhaps resulting in a lower rate of adverse reactions. Vancomycin is bactericidal against most susceptible organisms, but is bacteriostatic against the enterococcus. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus. Vancomycin attains good tissue and bone penetration and must be administered intravenously for systemic infections. It is eliminated renally with a half-life of about 6 hours; this half-life is prolonged with advanced age and reduced renal function. Significant adverse reactions can be minimized with careful attention to administration technique. Metronidazole has excellent tissue penetration; its antibacterial site of action is within anaerobic bacterial cells. Metronidazole is cleared by hepatic metabolism with a half-life of about 7 to 8 hours. The half-life is unchanged with renal dysfunction but is prolonged in patients with hepatic function impairment. Although adverse effects are relatively minor, there is an important interaction with warfarin. For podiatric infections, metronidazole can be used in skin- and soft-tissue infections; anaerobes in bone and joint infections are rare. Topical metronidazole has been used successfully in the treatment of decubitus ulcers, and this needs further evaluation. The most commonly used tetracyclines are tetracycline, doxycycline, and minocycline. The tetracyclines are broad spectrum antimicrobial agents, but their usefulness is limited by resistant strains. Tetracycline's absorption is significantly impaired by food, and it is cleared renally and fecally. Doxycycline has the highest protein binding and the longest half-life; it is cleared both renally and fecally without hepatic metabolism. Minocycline has the best absorption and tissue penetration; the unchanged drug is cleared renally and fecally, and it also undergoes hepatic metabolism. All tetracyclines have important adverse reactions with respect to teeth and bones, and they are contraindicated during pregnancy and for children under age eight.

A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function.

In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro. Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag. The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4. We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein. Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis. These and related results suggest that the amino-terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure. The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed.

The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT alpha 2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha 2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha 2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha 2 degradation is dependent on its ubiquitination.

Antibiotic therapy for common infections.

Several important points regarding the treatment of urinary tract infections should be made. Single-dose and short-course antibiotic therapy is appropriate only for women with acute bacterial cystitis due to E. coli. Studies comparing single-dose to full-course therapy have not been sufficiently designed to draw valid statistical conclusions, and only TMP/SMX is recommended at this time. Recurrent UTI in women is almost always due to reinfection, which is best managed by prophylactic antibiotics. Acute bronchitis and acute exacerbations of chronic bronchitis are often due to viral infections, and therefore antibiotic therapy is not always needed. In acute exacerbations of chronic bronchitis, the clearest success rates for antibiotic therapy have been in patients, who have all three of the following symptoms: increased dyspnea, increased sputum production, and sputum purulence. Mupirocin is an important addition to the agents used to treat bacterial skin infections due to streptococcal and staphylococcal strains. In impetigo, mupirocin has been demonstrated to be as effective or superior to oral erythromycin. In prostatitis, data on the fluoroquinolones appears impressive, but further comparative trials are needed. They may become first-line, empiric therapy. The newer oral antibiotics are not recommended as initial, empiric therapy in the outpatient management of common infections, with the possible exception of the treatment of prostatitis. These newer agents may be more important in the treatment of recurrent or resistant infections.

In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes.

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is discussed.

The obtention of simian virus 40 recombinants carrying d(CG.GC)n, d(CA.GT)n and d(CT.GA)n sequences. Stability of the inserted simple repeating sequences.

A general strategy for the introduction of simple repeating DNA sequences into the simian virus 40 (SV40) has been developed. SV40 recombinants carrying d(CG.GC)5, d(CA.GT)30 or d(CT.GA)22 insertions at either the TaqI site (position 4739) or the HpaII site (position 346) were obtained and the stability of the inserted DNA sequences studied. The palindromic potentially Z-DNA-forming d(CG.GC)n sequence was found to be highly unstable when compared to either d(CA.GT)n or d(CT.GA)n.

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences.

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

DNA conformation and chromatin organization of a d(CA/GT)30 sequence cloned in SV40 minichromosomes.

The alternating DNA sequence d(CA/GT)n is known to adopt the left-handed Z-form in negatively supercoiled DNA in vitro. This element represents a significant fraction of the highly repeated DNA sequences found in eukaryotic genomes. We have cloned an alternating d(CA/GT)30 sequence into SV40 minichromosomes at the unique HpaII restriction site which occurs in the transcriptional leader region of the viral late genes. By comparing the linkage differences of topoisomers obtained from viral DNA with or without the d(CA/GT)30 insert, at various stages of the lytic cycle, we conclude that this sequence does not predominate in the Z-form in vivo. Furthermore, we find that the d(CA/GT)30 sequence is packaged into nucleosomal core particles and the region of the minichromosomes which contain the d(CA/GT)30 sequence is organized as nucleosomes.

Internal structure of discrete nucleohistone complexes which form in vitro under conditions of physiological ionic strength.

Three discrete histone-DNA complexes assemble spontaneously when the four core histones are mixed with DNA under conditions which are close to physiological (0.15 M NaCl, pH 8). These species include the (H2A,H2B) dimeric complex (P1), the (H2A2,H2B2,H3,H4) hexameric complex (P2) and the nucleosome core complex (P3). This report compares several properties of these complexes with the properties of nucleosome cores assembled at high ionic strength (0.6 M NaCl). Based on histone-histone cross-linking studies, CD spectra, and thermal denaturation experiments, P1 is structurally similar to the subnucleosomal (H2A,H2B) fragment isolated from nuclease-digested chromatin. P1, P2, P3, and high salt-assembled nucleosome cores can all incorporate (H2A,H2B) pairs which have been previously cross-linked. Although the CD spectra and thermal denaturation profiles of P2 and P3 are closely related to those of nucleosome cores assembled in 0.6 M NaCl, cross-linking studies indicate that the arrangement of the histones in P2, and in a proportion of the P3 particles assembled in 0.15 M NaCl, are significantly different from their arrangement in nucleosome cores assembled in 0.6 M NaCl. The single cysteine residue on the H3 of P2 is accessible to the solvent. The two fluorescently labeled cysteine residues in a large proportion of the P3 particles assembled in 0.15 M NaCl are in a different orientation with respect to each other than the same residues in nucleosome cores assembled at high ionic strength.

Pyrimidine dimers induced in Escherichia coli DNA by ultraviolet radiation present in sunlight.

Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine-thymine dimers (C mean value of T) were more frequent than thymine dimers (T mean value of T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295-315 nm), C mean value of T are probably more important than T mean value of T.