Search PubMed⌕ Search

Biomedical subjects

Lawrence A Loeb

Publications and source records attributed to Lawrence A Loeb.

At least 19 recordsLinked to original sources

Human cancers express a mutator phenotype.

Cancer cells contain numerous clonal mutations, i.e., mutations that are present in most or all malignant cells of a tumor and have presumably been selected because they confer a proliferative advantage. An important question is whether cancer cells also contain a large number of random mutations, i.e., randomly distributed unselected mutations that occur in only one or a few cells of a tumor. Such random mutations could contribute to the morphologic and functional heterogeneity of cancers and include mutations that confer resistance to therapy. We have postulated that malignant cells exhibit a mutator phenotype resulting in the generation of random mutations throughout the genome. We have recently developed an assay to quantify random mutations in human tissue with unprecedented sensitivity. Here, we report measurements of random single-nucleotide substitutions in normal and neoplastic human tissues. In normal tissues, the frequency of spontaneous random mutations is exceedingly low, less than 1 x 10(-8) per base pair. In contrast, tumors from the same individuals exhibited an average frequency of 210 x 10(-8) per base pair, an elevation of at least two orders of magnitude. Our data document tumor heterogeneity at the single-nucleotide level, indicate that accelerated mutagenesis prevails late into tumor progression, and suggest that elevation of random mutation frequency in tumors might serve as a novel prognostic indicator.

DNA, Single-Stranded↗

Mutations in DNA polymerase eta are not detected in squamous cell carcinoma of the skin.

The major etiological agent in skin cancer is exposure to UV-irradiation and the concomitant DNA damage. UV-induced DNA lesions, such as thymine dimers, block DNA synthesis by the major DNA polymerases and inhibit the progression of DNA replication. Bypass of thymine dimers and related lesions is dependent on the translesion polymerase DNA polymerase eta (Poleta). In the inherited disorder, xeroderma pigmentosum variant (XPV), inactivation of Poleta results in extreme sensitivity to UV light and a marked increase in the incidence of skin cancer. Here, we tested the hypothesis that somatic mutations and/or polymorphisms in the POLH gene that encodes Poleta are associated with the induction of UV-dependent skin cancers. We sequenced the exonic regions of the Poleta open reading frame in DNA from 17 paired samples of squamous cell skin carcinoma and adjacent histologically normal tissue. We analyzed approximately 120,000 nucleotides and detected no mutations in POLH in the tumors. However, we identified 6 different single-nucleotide polymorphisms, 3 of them previously undocumented, which were present in both the tumor and paired normal tissue. We conclude that neither mutations nor polymorphisms in the coding regions of POLH are required for the generation of human skin squamous cell carcinoma.

Adult↗

Efficiency of carcinogenesis with and without a mutator mutation.

Carcinogenesis involves the acquisition of multiple genetic changes altering various cellular phenotypes. These changes occur within the fixed time period of a human lifespan, and mechanisms that accelerate this process are more likely to result in clinical cancers. Mutator mutations decrease genome stability and, hence, accelerate the accumulation of random mutations, including those in oncogenes and tumor suppressor genes. However, if the mutator mutation is not in itself oncogenic, acquiring that mutation would add an extra, potentially time-consuming step in carcinogenesis. We present a deterministic mathematical model that allows quantitative prediction of the efficiency of carcinogenesis with and without a mutator mutation occurring at any time point in the process. By focusing on the ratio of probabilities of pathways with and without mutator mutations within cell lineages, we can define the frequency or importance of mutator mutations in populations independently of absolute rates and circumvent the question of whether mutator mutations are "necessary" for cancers to evolve within a human lifetime. We analyze key parameters that predict the relative contribution of mutator mutants in carcinogenesis. Mechanisms of carcinogenesis involving mutator mutations are more likely if they occur early. Involvement of mutator mutations in carcinogenesis is favored by an increased initial mutation rate, by greater fold-increase in mutation rate due to the mutator mutation, by increased required steps in carcinogenesis, and by increased number of cell generations to the development of cancer.

Animals↗

Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells.

P140K-MGMT and G156A-MGMT genes encode two O(6)-benzylguanine-resistant O(6)-alkylguanine DNA alkyltransferase proteins that confer a high degree of O(6)-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O(6)-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O(6)-benzylguanine-resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O(6)-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED(50) for O(6)-benzylguanine, 60 micromol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED(50) for benzylguanine, >1,000 micromol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O(6)-benzylguanine (800 micromol/L) and temozolomide (1,000 micromol/L) or BCNU (20 micromol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O(6)-benzylguanine-resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection.

Alkylating Agents↗

Generation of mutator mutants during carcinogenesis.

Mutations are rare in normal cells. In contrast, multiple mutations are characteristic in most tumors. Previously we proposed a "mutator phenotype" hypothesis to explain how pre-cancer cells may acquire large number of mutations during carcinogenesis. Here we extend the "mutator phenotype" hypothesis considering recently discovered biochemical activities whose aberrant expression may result in genome-wide random mutations. The scope of this article is to emphasize that simple random point mutations can drive carcinogenesis and highlight new emerging pathways that generate these mutations. We focus specifically on random point mutations generated by replication errors, oxidative base damage, covalent base modifications by enzymes, and spontaneously generated abasic sites as a source of mutator mutants.

Animals↗

Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase delta.

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.

Amino Acid Sequence↗

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.

Amino Acid Motifs↗

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity.

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.

Animals↗

Current advances in unraveling the function of the Werner syndrome protein.

Werner syndrome (WS) is an autosomal recessive premature aging disease manifested by the mimicry of age-related phenotypes such as atherosclerosis, arteriosclerosis, cataracts, osteoporosis, soft tissue calcification, premature thinning, graying, and loss of hair, as well as a high incidence of some types of cancers. The gene product defective in WS, WRN, is a member of the RecQ family of DNA helicases that are widely distributed in nature and believed to play central roles in genomic stability of organisms ranging from prokaryotes to mammals. Interestingly, WRN is a bifunctional protein that is exceptional among RecQ helicases in that it also harbors an exonuclease activity. Furthermore, it preferentially operates on aberrant DNA structures believed to exist in vivo as intermediates in specific DNA transactions such as replication (forked DNA), recombination (Holliday junction, triplex and tetraplex DNA), and repair (partial duplex with single stranded bubble). In addition, WRN has been shown to physically and functionally interact with a variety of DNA-processing proteins, including those that are involved in resolving alternative DNA structures, repair DNA damage, and provide checkpoints for genomic stability. Despite significant research activity and considerable progress in understanding the biochemical and molecular genetic function of WRN, the in vivo molecular pathway(s) of WRN remain elusive. The following review focuses on the recent advances in the biochemistry of WRN and considers the putative in vivo functions of WRN in light of its many protein partners.

DNA Helicases↗

Negative clonal selection in tumor evolution.

Development of cancer requires the acquisition of multiple oncogenic mutations and selection of the malignant clone. Cancer evolves within a finite host lifetime and mechanisms of carcinogenesis that accelerate this process may be more likely to contribute to the development of clinical cancers. Mutator mutations are mutations that affect genome stability and accelerate the acquisition of oncogenic mutations. However, mutator mutations will also accelerate the accumulation of mutations that decrease cell proliferation, increase apoptosis, or affect other key fitness parameters. These "reduced-fitness" mutations may mediate "negative clonal selection," i.e., selective elimination of premalignant mutator clones. Target reduced-fitness loci may be "recessive" (both copies must be mutated to reduce fitness) or "dominant" (single-copy mutation reduces fitness). A direct mathematical analysis is applied to negative clonal selection, leading to the conclusion that negative clonal selection against mutator clones is unlikely to be a significant effect under realistic conditions. In addition, the relative importance of dominant and recessive reduced-fitness mutations is quantitatively defined. The relative predominance of mutator mutations in clinical cancers will depend on several variables, including the tolerance of the genome for reduced-fitness mutations, particularly the number and potency of dominant reduced-fitness loci.

Cell Lineage↗

Quantification of random genomic mutations.

Cancer cells contain numerous clonal mutations. It has been theorized that malignant cells sustain an elevated mutation rate and, as a consequence, harbor yet larger numbers of random point mutations. Testing this hypothesis has been precluded by lack of an assay to measure random mutations-that is, mutations that occur in only one or a few cells of a population. We have established a method that has permitted us to detect and identify rare random mutations in human cells, at a frequency of 1 per 10(8) base pairs. The assay is based on gene capture, by hybridization with a uracil-containing probe, followed by magnetic separation. Mutations that render the mutational target sequence non-cleavable by a restriction enzyme are quantified by dilution to single molecules and real-time quantitative PCR amplification. The assay can be extended to quantify mutation in any DNA-based organism, at different sites in the genome, in introns and exons, in unselected and selected genes, and in proliferating and quiescent cells.

Chromosome Mapping↗

Critical role of R-loops in processing replication blocks.

Blocks in replication result from impediments to the advancing replication machinery and are lethal if not resolved. The replication fork must be reassembled for DNA synthesis to proceed. Fork assembly outside the chromosomal origin of replication (oriC) is mediated by recombination or via a helicase-dependent pathway. ColE1 plasmid origins of replication and oriK sites initiate primosome assembly by an RNA-DNA hybrid structure known as R-loop. We review evidence suggesting that R-loops are frequent during normal cell growth and that R-loops are critical for the maintenance of genome integrity. We propose that downstream of a replication block, RNA at R-loops is extended by DNA polymerase I, opening up the DNA duplex and leading to the recruitment of the replisome. This would allow replication to proceed while the original block is repaired or bypassed. Unlike recombination and helicase-dependent fork restoration, this mechanism would operate preferentially in transcribed areas of the genome, which are known to be particularly susceptible to DNA damage. Our model emphasizes the intimate relationship between transcription and repair, offers a unifying interpretation of phenotypes attributed to bacterial strains deficient in R-loop fork assembly, and calls for a renewed focus on R-loop formation and regulation.

DNA↗

Genetic instability in cancer: theory and experiment.

Epidemiologic data and molecular biology have combined to demonstrate that multiple genetic changes may be required in carcinogenesis. Mutator mutations, defined as genetic changes which increase the rate of genetic change, including both single base changes and chromosomal instability, may accelerate this process. Key questions remain in defining the role of mutator mutations in carcinogenesis as well as in cancer therapy. Theoretical approaches, including deterministic and stochastic models, have played a significant role in hypothesis generation, experimental design, and refinement of conclusions in this field, and are expected to continue to do so in the future.

Cell Transformation, Neoplastic↗

Lethal mutagenesis of HIV.

HIV-1 and other retroviruses exhibit mutation rates that are 1,000,000-fold greater than their host organisms. Error-prone viral replication may place retroviruses and other RNA viruses near the threshold of "error catastrophe" or extinction due to an intolerable load of deleterious mutations. Strategies designed to drive viruses to error catastrophe have been applied to HIV-1 and a number of RNA viruses. Here, we review the concept of extinguishing HIV infection by "lethal mutagenesis" and consider the utility of this new approach in combination with conventional antiretroviral strategies.

APOBEC-3G Deaminase↗

Incorporation of reporter-labeled nucleotides by DNA polymerases.

The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

DNA↗

The enzymatic activities of the Werner syndrome protein are disabled by the amino acid polymorphism R834C.

The Werner syndrome protein, WRN, is a member of the RecQ family of DNA helicases. It possesses both 3'-->5' DNA helicase and 3'-->5' DNA exonuclease activities. Mutations in WRN are causally associated with a rare, recessive disorder, Werner syndrome (WS), distinguished by premature aging and genomic instability; all are reported to result in loss of protein expression. In addition to WS-linked mutations, single nucleotide polymorphisms, with frequencies that exceed those of WS-associated mutations, are also present in WRN. We have initiated studies to determine if six of these polymorphisms affect the enzymatic activities of WRN. We show that two common polymorphisms, F1074L and C1367R, and two infrequent polymorphisms, Q724L and S1079L, exhibit little change in activity relative to wild-type WRN; the polymorphism, T172P, shows a small but consistent reduction of activity. However, an infrequent polymorphism, R834C, located in the helicase domain dramatically reduces WRN helicase and helicase-coupled exonuclease activity. The structure of the E. coli helicase core suggests that R834 may be involved in interactions with ATP. As predicted, substitution of Arg with Cys interferes with ATP hydrolysis that is absolutely required for unwinding DNA. R834C thus represents the first missense amino acid polymorphism in WRN that nearly abolishes enzymatic activity while leaving expression largely unaffected.

Adenosine Triphosphatases↗