Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

A phenotype-based screen for embryonic lethal mutations in the mouse

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.

The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Overexpression of DNA polymerase β in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.

Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Phylogeny of mRNA capping enzymes

The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure–function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.

An intercalation inhibitor altering the target selectivity of DNA damaging agents: Synthesis of site-specific aflatoxin B1 adducts in a p53 mutational hotspot

Aflatoxin B1 (AFB1) is a potent human carcinogen implicated in the etiology of hepatocellular carcinoma. Upon metabolic activation to the reactive epoxide, AFB1 forms DNA adducts primarily at the N7 position of guanines. To elucidate more fully the molecular mechanism of AFB1-induced mutagenesis, an intercalation inhibitor was designed to probe the effects of intercalation by AFB1 epoxide on its reaction with DNA. DNA duplexes were prepared consisting of a target strand containing multiple potentially reactive guanines and a nontarget strand containing a cis-syn thymidine-benzofuran photoproduct. Because the covalently linked benzofuran moiety physically occupies an intercalation site, we reasoned that such a site would be rendered inaccessible to AFB1 epoxide. By strategic positioning of this intercalation inhibitor in the intercalation site 5′ to a specific guanine, the adduct yield at that site was greatly diminished, indicating that intercalation by AFB1 epoxide contributes favorably to adduct formation. Using this approach it has been possible to simplify the production of site-specifically modified oligonucleotides containing AFB1 adducts in the sequence context of a p53 mutational hotspot. Moreover, we report herein isolation of site-specifically AFB1-modified oligonucleotides in sequences containing multiple guanines. Use of intercalation inhibitors will facilitate both investigation of the ability of other carcinogens to intercalate into DNA and the synthesis of specific carcinogen-DNA adducts.

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH → Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.

Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens

Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10–100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at −50°C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety.