Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts.Trial RegistrationClinicalTrials.gov NCT01087307
Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minusstrand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was >10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus-and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.The causative agent of AIDS, human immunodeficiency virus type 1 (HIV-1), exhibits extensive genomic heterogeneity (1). One source of this sequence diversity is inaccurate DNA replication by its reverse transcriptase (RT). Conversion of the viral genomic RNA to double-stranded DNA is a two-step process. The minus strand is synthesized by using viral RNA as a template. The RT then uses this newly synthesized, complementary DNA as a template for secondstrand synthesis.HIV-1 RT, which lacks a 3'--5' exonuclease proofreading activity (2), has been shown to be error-prone during DNAdependent DNA synthesis in vitro (2-7). Although such studies are relevant to second-strand synthesis, the fidelity of reverse transcription of RNA to DNA is also relevant to retroviral mutagenesis. Limited information is available on the fidelity of RNA-dependent DNA synthesis by HIV-1 RT (8-10). We therefore decided to examine HIV-1 RT fidelity with heteropolymeric RNA by adapting a forward mutation assay (2) previously used to establish HIV-1 RT error rates with a DNA template (4). This assay has permitted a direct comparison of fidelity of HIV-1 RT with RNA and DNA templates of the same sequence as well as an estimate for error rates during transcription by T7 RNA polymerase. MATERIALS AND METHODSEnzymes and Reagents. The recombinant form o...
An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5' or 3' to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type hMLH1 gene and is deficient in repair of mismatches. However, a cell line with deletions in both hMSH2 alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.
Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.genetic instability ͉ microsatellite ͉ mutation rate ͉ Saccharomyces cerevisiae C omparisons of amino acid or base sequences of orthologous genes indicate that different genes evolve at different rates (1). Differences in the rates of accumulation of amino acid changes or nonsynonymous base substitutions are influenced by selective constraints (1). For highly expressed genes, the rate of synonymous base substitutions is affected by GC content and codon bias (2, 3). In Saccharomyces cerevisiae and mammalian cells, the rates of synonymous substitutions also vary by a factor of Ϸ10, depending on the position of the gene in the genome (4-6). The interpretation of these observations is unclear. It is possible that the misincorporation rates of the replicative DNA polymerases are different at different positions in the genome. Alternatively, the fidelity of DNA polymerases may be invariant, but the detection and repair of misincorporation events may be context-specific.Microsatellites are regions of DNA in which a single base or a small number of bases is repeated in tandem. The polyGT sequence is a particularly common microsatellite in many eukaryotes (7). We have developed (8, 9) methods of measuring the rate of microsatellite alterations. In this study, we used this assay to measure the mutation rates of the same polyGT microsatellite placed in 10 different chromosomal contexts in the yeast genome. We show that the microsatellite mutation rates vary by more than an order of magnitude among different genomic positions in yeast strains that have wild-type DNA mismatch repair. We have demonstrated (8) that the mutation rates of microsatellites are greatly elevated in yeast strains with deficient mismatch repair. In this study, we find that microsatellite in...
We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.