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Biomedical subjects

Lawrence A Loeb

Publications and source records attributed to Lawrence A Loeb.

At least 37 records · Page 2Linked to original sources

Destabilization of tetraplex structures of the fragile X repeat sequence (CGG)n is mediated by homolog-conserved domains in three members of the hnRNP family.

Hairpin or tetrahelical structures formed by a d(CGG)n sequence in the FMR1 gene are thought to promote expansion of the repeat tract. Subsequent to this expansion FMR1 is silenced and fragile X syndrome ensues. The injurious effects of d(CGG)n secondary structures may potentially be countered by agents that act to decrease their stability. We showed previously that the hnRNP-related protein CBF-A destabilized G'2 bimolecular tetraplex structures of d(CGG)n. Analysis of mutant proteins revealed that the CBF-A-conserved domains RNP11 and ATP/GTP binding box were sufficient and necessary for G'2 d(CGG)n disruption while the RNP21 motif inhibited the destabilization activity. Here, we report that a C-terminal fragment of CBF-A whose only remaining conserved domain was the ATP/GTP binding motif, disrupted G'2 d(CGG)n more selectively than wild-type CBF-A. Further, two additional members of the hnRNP family, hnRNP A2 and mutant hnRNP A1 effectively destabilized G'2 d(CGG)n. Examination of mutant hnRNP A2 proteins revealed that, similar to CBF-A, their RNP11 element and ATP/GTP binding motif mediated G'2 d(CGG)n disruption, while the RNP21 element blocked their action. Similarly, the RNP11 and RNP21 domains of hnRNP A1 were, respectively, positive and negative mediators of G'2 d(CGG)n destabilization. Last, employing the same conserved motifs that mediated disruption of the DNA tetraplex G'2 d(CGG)n, hnRNP A2 destabilized r(CGG)n RNA tetraplex.

Adenosine Triphosphate↗

Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea.

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.

Antineoplastic Agents↗

Protein tolerance to random amino acid change.

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10(5) diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.

Amino Acid Substitution↗

The Werner syndrome protein confers resistance to the DNA lesions N3-methyladenine and O6-methylguanine: implications for WRN function.

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O(6)-methylguanine adducts as well, but only when repair by O(6)-methylguanine-DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents.

4-Nitroquinoline-1-oxide↗

Environmental and chemical carcinogenesis.

People are continuously exposed exogenously to varying amounts of chemicals that have been shown to have carcinogenic or mutagenic properties in experimental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. It has been estimated that exposure to environmental chemical carcinogens may contribute significantly to the causation of a sizable fraction, perhaps a majority, of human cancers, when exposures are related to "life-style" factors such as diet, tobacco use, etc. This chapter summarizes several aspects of environmental chemical carcinogenesis that have been extensively studied and illustrates the power of mechanistic investigation combined with molecular epidemiologic approaches in establishing causative linkages between environmental exposures and increased cancer risks. A causative relationship between exposure to aflatoxin, a strongly carcinogenic mold-produced contaminant of dietary staples in Asia and Africa, and elevated risk for primary liver cancer has been demonstrated through the application of well-validated biomarkers in molecular epidemiology. These studies have also identified a striking synergistic interaction between aflatoxin and hepatitis B virus infection in elevating liver cancer risk. Use of tobacco products provides a clear example of cancer causation by a life-style factor involving carcinogen exposure. Tobacco carcinogens and their DNA adducts are central to cancer induction by tobacco products, and the contribution of specific tobacco carcinogens (e.g. PAH and NNK) to tobacco-induced lung cancer, can be evaluated by a weight of evidence approach. Factors considered include presence in tobacco products, carcinogenicity in laboratory animals, human uptake, metabolism and adduct formation, possible role in causing molecular changes in oncogenes or suppressor genes, and other relevant data. This approach can be applied to evaluation of other environmental carcinogens, and the evaluations would be markedly facilitated by prospective epidemiologic studies incorporating phenotypic carcinogen-specific biomarkers. Heterocyclic amines represent an important class of carcinogens in foods. They are mutagens and carcinogens at numerous organ sites in experimental animals, are produced when meats are heated above 180 degrees C for long periods. Four of these compounds can consistently be identified in well-done meat products from the North American diet, and although a causal linkage has not been established, a majority of epidemiology studies have linked consumption of well-done meat products to cancer of the colon, breast and stomach. Studies employing molecular biomarkers suggest that individuals may differ in their susceptibility to these carcinogens, and genetic polymorphisms may contribute to this variability. Heterocyclic amines, like most other chemical carcinogens, are not carcinogenic per se but must be metabolized by a family of cytochrome P450 enzymes to chemically reactive electrophiles prior to reacting with DNA to initiate a carcinogenic response. These same cytochrome P450 enzymes--as well as enzymes that act on the metabolic products of the cytochromes P450 (e.g. glucuronyl transferase, glutathione S-transferase and others)--also metabolize chemicals by inactivation pathways, and the relative amounts of activation and detoxification will determine whether a chemical is carcinogenic. Because both genetic and environmental factors influence the levels of enzymes that metabolically activate and detoxify chemicals, they can also influence carcinogenic risk. Many of the phenotypes of cancer cells can be the result of mutations, i.e., changes in the nucleotide sequence of DNA that accumulate as tumors progress. These can arise as a result of DNA damage or by the incorporation of non-complementary nucleotides during DNA synthetic processes. Based upon the disparity between the infrequency of spontaneous mutations and the large numbers of mutations reported in human tumors, it has been postulated that cancers must exhibit a mutator phenotype, which would represent an early event in cancer progression. A mutator phenotype could be generated by mutations in genes that normally function to guarantee genetic stability. These mutations presumably arise via DNA damage by environmental or endogenous agents, but it remains to be determined whether the acquisition of a mutator phenotype is a necessary event during tumor progression.

Aflatoxins↗

Viral error catastrophe by mutagenic nucleosides.

Riboviruses and retroviruses have the highest rates of mutations of any known organism. Increasing the mutation rate of these viruses could exceed the error threshold for viability of a viral population within a host. Recent experiments with mutagenic nucleoside analogs validate this new approach to treating infection of RNA viruses. Lethal mutagenesis with HIV-infected cells in culture has been documented and has been postulated to be the mechanism for treatment of hepatitis C with ribavirin. We consider the viral dynamics involved in the formation of a quasispecies, the choice of mutagenic nucleoside analogs, and the studies that have demonstrated the feasibility of lethal mutagenesis.

Antiviral Agents↗

When pol I goes into high gear: processive DNA synthesis by pol I in the cell.

Pol I is the most abundant polymerase in E. coli and plays an important role in short patch repair. In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro. Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication. In a previous publication, we described the generation of a highly error-prone DNA polymerase I. Expression of this mutant Pol I results in errors during the replication of a ColE1 plasmid. The distribution and spectrum of mutations in the ColE1 plasmid sequence downstream the ori indicates that Pol I is capable of more processive replication in vivo than previously accepted. Here, we review evidence suggesting that Pol I may be recruited into a replisome-like holoenzyme and speculate that processive DNA replication by Pol I may play a role in recombination-dependent DNA replication in the cell.

Chromosomes↗

Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site.

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.

Amino Acid Sequence↗

Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase.

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20-50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq DNA pol/TBD is thermostable, PCR competent and able to copy repetitive deoxynucleotide sequences six to seven times more faithfully than Taq DNA polymerase and makes 2-3-fold fewer AT-->GC transition mutations.

Amino Acid Sequence↗

Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.

We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.

Anti-Bacterial Agents↗

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta.

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.

Binding Sites↗

Multiple mutations and cancer.

Most human tumors are highly heterogenous. We have hypothesized that this heterogeneity results from a mutator phenotype. Our premise is that normal mutation rates are insufficient to account for the multiple mutations found in human cancers, and, instead, that cancers must exhibit a mutator phenotype early during their evolution. Here, we examine the current status and implications of the mutator phenotype hypothesis for the prognosis, treatment, and prevention of human cancers.

Base Sequence↗

Tumbling down a different pathway to genetic instability.

Ulcerative colitis (UC), a chronic inflammatory condition associated with a predisposition to colon cancer, is frequently characterized by DNA damage in the form of microsatellite instability (MSI). A new report links inflammation in UC with increases in the DNA repair enzymes 3-methyladenine DNA glycosylase and apurinic/apyrimidinic endonuclease, and, paradoxically, with increased MSI. These findings may represent a novel mechanism contributing to MSI in chronic inflammation.

Base Sequence↗