A high-resolution landscape of mutations in the super-enhancer in normal human B cells.

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities.

Tumor necrosis factor, tumor necrosis factor inhibition, and cancer risk.

Objective: Tumor necrosis factor (TNF) is a highly pleiotropic cytokine with multiple activities other than its originally discovered role of tumor necrosis in rodents. TNF is now understood to play a contextual role in driving either tumor elimination or promotion. Using both animal and human data, this review examines the role of TNF in cancer development and the effect of TNF and TNF inhibitors (TNFis) on malignancy risk.

DNA replication error-induced extinction of diploid yeast.

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown.

Emergence of DNA polymerase ε antimutators that escape error-induced extinction in yeast.

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system.

Decoding cell lineage from acquired mutations using arbitrary deep sequencing.

Because mutations are inevitable, the genome of each cell in a multicellular organism becomes unique and therefore encodes a record of its ancestry. Here we coupled arbitrary single primer PCR with next-generation DNA sequencing to catalog mutations and deconvolve the phylogeny of cultured mouse cells. This study helps pave the way toward construction of retrospective cell-fate maps based on mutations accumulating in genomes of somatic cells.

Mutator suppression and escape from replication error-induced extinction in yeast.

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold.

Antimutator variants of DNA polymerases.

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates.

A random mutation capture assay to detect genomic point mutations in mouse tissue.

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain.

DNA replication fidelity and cancer.

Cancer is fueled by mutations and driven by adaptive selection. Normal cells avoid deleterious mutations by replicating their genomes with extraordinary accuracy. Here we review the pathways governing DNA replication fidelity and discuss evidence implicating replication errors (point mutation instability or PIN) in carcinogenesis.

Case series: acute tumor lysis syndrome in mutator mice with disseminated lymphoblastic lymphoma.

Acute tumor lysis syndrome (ATLS) is characterized by severe metabolic abnormalities and organ dysfunction resulting from rapid destruction of neoplastic cells. Metabolic disturbances are thought to be the primary cause of clinical ATLS symptoms, which include renal dysfunction, seizures, and cardiac arrhythmias. The histopathologic lesions associated with organ dysfunction are largely unknown because of the low rate of mortality of ATLS in humans and the few cases of ATLS identified in laboratory animals.

Divergent cellular phenotypes of human and mouse cells lacking the Werner syndrome RecQ helicase.

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn-/- mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn-/- mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts.

DNA polymerase epsilon and delta proofreading suppress discrete mutator and cancer phenotypes in mice.

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols epsilon and delta, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown.

Antiretroviral drug resistance in HIV-2: three amino acid changes are sufficient for classwide nucleoside analogue resistance.

Genotypic surveys suggest that human immunodeficiency virus type 1 (HIV-1) and HIV-2 evolve different sets of mutations in response to nucleoside reverse-transcriptase inhibitors (NRTIs). We used site-directed mutagenesis, culture-based phenotyping, and cell-free assays to determine the resistance profiles conferred by specific amino acid replacements in HIV-2 reverse transcriptase. Although thymidine analogue mutations had no effect on zidovudine sensitivity, the addition of Q151M together with K65R or M184V was sufficient for high-level resistance to both lamivudine and zidovudine in HIV-2, and the combination of K65R, Q151M, and M184V conferred classwide NRTI resistance.

Human immunodeficiency virus types 1 and 2 exhibit comparable sensitivities to Zidovudine and other nucleoside analog inhibitors in vitro.

Using an indicator cell assay that directly quantifies viral replication, we show that human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) exhibit similar sensitivities to 3'-azido-3'-deoxythymidine (zidovudine) as well as other nucleoside analog inhibitors of reverse transcriptase. These data support the use of nucleoside analogs for antiviral therapy of HIV-2 infection.

Mutation at the polymerase active site of mouse DNA polymerase delta increases genomic instability and accelerates tumorigenesis.

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta.

DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions.

Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture.

DNA replication fidelity: proofreading in trans.

Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.

Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase.

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction.

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.

Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage.

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired.

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.

Purifying selection masks the mutational flexibility of HIV-1 reverse transcriptase.

DNA and RNA polymerases share a core architecture composed of three structurally conserved motifs: A, B, and C. Although the amino acid sequences of these motifs are highly conserved between closely related organisms, variation across broader evolutionary distances suggests that only a few residues in each motif are indispensable for polymerase function. To test this, we constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function.

Ig gene somatic hypermutation in mice defective for DNA polymerase delta proofreading.

This study is an investigation of the possible role of DNA polymerase (pol) delta with an inactivated exonuclease (exo) in somatic hypermutation (SHM). Analysis of endogenous heavy chain transcripts revealed no difference in mutation frequency and pattern between exo(-/-), exo(+/-) and exo(+/+) mice. The lack of an effect of the pol delta exo mutation on SHM could be due to: (i) normally pol delta is used in SHM, but the exo is prevented from proofreading, (ii) normally pol delta is used, but the decrease in fidelity of the exo(-) pol does not increase hypermutation frequency enough to be detected, and (iii) pol delta is not used in SHM.

Incorporation of uracil into minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription.

Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture.