Mapping of sites on the surface membrane of mammalian cells

Summary Cells transformed by Simian Virus 40 have sites on the surface membrane for Concanavalin A (Con A) and a copolymer of ornithine, leucine (POL). The cells can be rapidly agglutinated by Con A, more slowly aggregated by POL, and they can be killed by both compounds. Treatment with Con A or POL has been used to select resistant cell variants from the transformed cells. Variants selected for resistance to Con A were also resistant to POL, but variants selected for resistance to POL were not resistant to Con A. The POL-selected variants showed less aggregation by POL but no decrease in agglutinability by Con A, whereas Con A-selected variants showed a decrease both in POL aggregation and Con A agglutination. The selection for both sites by Con A and only for POL sites by POL, can be explained in that the sites for POL are part of the sites for Con A and/or are included in clusters of Con A sites.Paper I in this series is Inbar, Ben-Bassat and Sachs (1971a).     

Repair of DNA-protein cross-links in mammalian cells

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).     

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.     

Defective replication of psoralen adducts detected at the gene-specific level in xeroderma pigmentosum variant cells.

Replication of damaged DNA is suspected to play an important role in cell cycle, genetic stability, and survival pathways. Using psoralen photoaddition as prototype DNA damage and the renaturing agarose gel electrophoresis technique to measure DNA cross-linking in individual genes, Vos and Hanawalt previously observed efficient bypass replication of psoralen monoadducts in human genes (J.-M. H. Vos and P. C. Hanawalt, Cell 50:789-799, 1987). To understand the mechanism of bypass replication in human cells, mutants affected in such a process would be useful. We now report that cells from individuals suffering from the hereditary recessive syndrome xeroderma pigmentosum variant (XPV) are hypersensitive to killing induced by photoactivated psoralen. In addition, analysis of psoralen-mediated DNA cross-linking in the rRNA genes indicated that although repair of psoralen adducts was similar to that of normal individuals, XPV cells were markedly deficient in the ability to bypass psoralen adducts during replication; in comparison with normal cells, approximately half as many monoadducts were bypassed during replication in XPV cells. Furthermore, in contrast to normal cells, replication of interstrand cross-links was not detected in XPV. This is the first demonstration of a deficiency in bypass replication detected at the gene-specific level in vivo. A model involving a strand-specific defect in recombinational bypass in XPV is proposed.     

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl_CUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.     

Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain

This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits. To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous. In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles.     

Sequence context at human single nucleotide polymorphisms: overrepresentation of CpG dinucleotide at polymorphic sites and suppression of variation in CpG islands

Human polymorphisms originate as mutations, and the influence of context on mutagenesis should be reflected in the distribution of sequences surrounding single nucleotide polymorphisms (SNPs). We have performed a computational survey of nearly two million human SNPs to determine if sequence-dependent hotspots for polymorphism exist in the human genome. Here we show that sequences containing CpG dinucleotides, which occur at low frequencies in the human genome, are 6.7-fold more abundant at polymorphic sites than expected. In contrast, polymorphisms in CpG sequences located within CpG islands, important regulatory regions that modulate gene expression, are 6.8-fold less prevalent than expected. The distribution of polymorphic alleles at CpGs in CpG islands is also significantly different from that in non-island regions. These data strongly support a role for 5-methylcytosine deamination in the generation of human variation, and suggest that variation at CpGs in islands is suppressed.     

Mapping at the nucleotide level of Xenopus laevis mitochondrial D-loop H strand: structural features of the 3' region

The D-loop resulting from limited synthesis of the newly replicated heavy (H) strand of mitochondrial DNA provides a good opportunity to examine both the origin and termination of DNA synthesis. We report here the precise determination of the 3' and 5' termini of nascent Xenopus laevis D-loop H strand. We observe two major classes of newly synthesized D-loop H strands, 1641 and 1675 nucleotides long. A stable putative secondary structure located around its 3' end is described. Analogous secondary structures are also found in the same region of the mammalian D-loop mitochondrial DNAs. Moreover a pentanucleotide (5' TACAT 3'), base-paired in these secondary structures and most often present in two copies, is conserved in all vertebrate species so far studied. The termination associated sequence previously described in mammals is part of the putative stop signal represented by the secondary structure except in man. These results show that the mechanism of arrest of H strand synthesis is common to vertebrates.     

Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).     

hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross-links in vitro

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSbeta but is not dependent upon the hMutSalpha complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSbeta complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.     

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry, toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of a replication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the action of structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. This process generates a double-strand break, which must be subsequently repaired by homologous recombination. Recent work provided evidence for a new, incision-independent unhooking mechanism involving intrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic site or the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair reverts to the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes might participate in the unhooking of chemically diverse interstrand DNA cross-links.     

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]- -tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.