Tolerance Of Human Cells Research Articles

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

Melanocyte-specific CD8+ T cells demonstrate tolerance characterized by an impaired ability to differentiate into a highly proliferative population

Abstract The mechanisms governing non-deletional CD8+ T cell tolerance are poorly understood. We are using a physiologically-relevant mouse model to better understand the regulation of self-specific CD8+ T cells, which impact both autoimmune disease and cancer. We find a substantial number of polyclonal cells specific for an epitope from the melanocyte/melanoma-associated enzyme tyrosinase-related protein 2 (Trp2) in both wild-type (WT) C57BL/6 and Trp2-deficient (Trp2 KO) mice. In pre-immune mice, these cells have a naïve phenotype in both strains, and they respond similarly for the first several days after in vivo stimulation with Trp2 peptide alone or in combination with adjuvants (TriVax). However, significantly fewer WT Trp2-specific cells are present at an effector timepoint after TriVax or recombinant Trp2-expressing Listeria monocytogenes relative to KO Trp2-specific cells. WT Trp2-specific cells also show a deficiency in mediating vitiligo compared with KO cells when TriVax-generated effectors are transferred into recipient WT mice primed with TriVax and dinitrofluorobenzene (DNFB). Single-cell RNA sequencing at day three after stimulation reveals that KO Trp2-specific cells efficiently differentiate into a proliferative population, whereas few WT Trp2-specific cells demonstrate this phenotype. As a population, WT Trp2-specific CD8+ T cells exhibit covert anergy: despite a naïve appearance at steady state and initial acquisition of activation markers after stimulation, these self-specific cells are unable to expand productively or to efficiently mediate functional anti-melanocyte activity. These findings have implications for recently-described non-deletional CD8+ T cell tolerance in humans.

Tunable Composition of Dynamic Non-Viral Vectors over the DNA Polyplex Formation and Nucleic Acid Transfection.

Polyethylene glycol (PEG) functionalization of non-viral vectors represents a powerful tool through the formation of an overall surface charge shielding ability, which is fundamental for efficient nucleic acid delivery systems. The degree of non-viral vector PEGylation and the molecular weight of utilized PEG is crucial since the excessive use of PEG units may lead to a considerable reduction of the DNA-binding capacity and, subsequently, in a reduction of in vitro transfection efficiency. Herein, we report a detailed study on a series of dynamic combinatorial frameworks (DCFs) containing PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) of different lengths, and branched low molecular weight polyethylenimine components, reversibly connected in hyperbranched structures, as efficient dynamic non-viral vectors. The obtained frameworks were capable of forming distinct supramolecular amphiphilic architectures, shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS), with sizes and stability depending on the length of PEG units. The interaction of PEGylated DCFs with nucleic acids was investigated by agarose gel retardation assay and atomic force microscopy (AFM), while their transfection efficiency (using pCS2+MT-Luc DNA as a reporter gene) and cytotoxicity were evaluated in HeLa cells. In addition, the data on the influence of the poly-(ethyleneglycol)-bis(3-aminopropyl) length in composition of designed frameworks over transfection efficiency and tolerance in human cells were analyzed and compared.

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells.

Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.

Activation Induced Cytidine Deaminase (Aid) Acts As a Gate Keeper in Pro-B Cells and Prevents PB-ALL

Introduction: Activation induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination in splenic germinal center B cells and is implicated in retaining central B cell tolerance in the bone marrow (BM) (Cantaert et al., Immunity, 2015). Moreover, there is recent in vitro evidence that AID is upregulated in precursor B cells after exposure to LPS, contributing to the clonal evolution of pB-ALL (Swaminathan et al., Nat Immunol, 2015) (Greaves M. and Müschen M., Cancer Discovery, 2015). These studies were carried out in pre-BII / early immature B cells, which are the first B cell compartments with detectable intrinsic AID expression. However a functional role of AID in pro-B cells is still controversially discussed and a functional role of AID in leukemogenesis remains speculative. We designed an in vivo model which allowed us the investigation of intrinsic Aid expression in tumor prone pro-B cells. Our data indicate that Aid is a gate keeper at the early stage of B cell development and its loss of function facilitates the development of pB-ALL.Methods: We crossed a Rag1 deficient tumor prone mouse model (p19Arf-/-/Rag1-/-) (Hauer et al., Blood, 2011) on an Aid deficient background to obtain Aid knockout (p19Arf-/-/Rag1-/-/Aid-/-) and heterozygous (p19Arf-/-/Rag1-/-/Aid+/-) mice. Healthy and diseased mice were characterized by immunohistochemistry, Flow cytometric analysis, genome and transcriptome profiling. Cell cycle analysis was performed with pro-B cells of healthy mice.Results: P19Arf-/-Rag1-/- mice display a B cell developmental arrest at the pro-B stage and develop pB-ALL at an incidence of 26 %. Surprisingly, an additional loss of Aid in these cells accelerated the pB-ALL incidence to 98 % (44/45, median onset 25 weeks). Moreover our model reproduces the dose dependent effect of AID on regulating B cell tolerance in humans, since Aid+/- mice on the same background displayed significant disease reduction (83 %, 15/18, median onset 33 weeks, Mantel-Cox Test p=0.0175). The leukemia displayed a pro-B cell phenotype (CD19+B220+ckit+IgM-) and manifested with splenomegaly, dissemination of blast cells to the BM, peripheral blood (PB) and spleen. Pro-B tumors from p19Arf-/-Rag1-/- mice expressed Aid on transcript (qRT-PCR) and protein (western blot) level, indicating that Aid expression is not restricted to CD19+ BM cells with co-expression of a functional IgM heavy chain product but rather occurs at earlier stages of B cell development. Again this effect was dose dependent, since in pB-ALLs of p19Arf-/-Rag1-/-Aid-/+ mice Aid expression was significantly reduced. To identify the second hit we performed whole exome sequencing of murine tumors, which revealed accumulation of recurrent somatic Jak3 (R653H, V670A) and Dnm2 (G397R) mutations. To extend these findings further, Sanger sequencing of these regions displayed a mutational pattern of somatic Jak3 mutations in 60 % of Aid+/- and 80 % of Aid-/-pB-ALLs, while Dnm2 was somatically mutated in 96 % of all pB-ALLs analyzed. The detected Jak3 variants are known to induce a constitutive active downstream signaling. Loss of function mutations in DNM2 can increase the IL-7R cell surface expression, which highlight the relevance of the IL7R signaling in the context of tumor progression. However we did not observe detectable Aid expression in healthy pro-B cells of p19Arf-/-Rag1-/- animals in line with findings from Cantaert et al. On the other hand loss of Aid expression accelerates the repopulation capacity starting at the pro-B cell compartment (Kuraoka et al., Proc Natl Acad Sci, 2011). In our model Aid loss produces a dose dependent increase in proliferation and BrdU assays of B220+ sorted pro-B cells of healthy mice from the different cohorts (30 % cells in S-Phase in p19Arf-/-Rag1-/-compared to 50 % S-Phase with additional Aid loss), although Aid expression is below the detection limit.Conclusion: We present in vivo evidence that Aid has a gate keeper function in pro-B cells, which allows aberrant IL-7 dependent pro-B cells without a functional receptor to be eliminated through Aid induction. This further extends the observation that Aid mediates the clearance of autoreactive early immature B-cell clones and is required to prevent pB-ALL. In this regard Aid overexpression but also loss of Aid expression can facilitate pB-ALL development. DisclosuresNo relevant conflicts of interest to declare.

280. Lentiviral-Mediated Gene Therapy Restores B Cell Homeostasis and Tolerance in Wiskott-Aldrich Syndrome Patients

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency characterized by micro-thrombocytopenia, eczema and increased risk of infections, autoimmunity and tumors. Allogeneic hematopoietic stem cell (HSC) transplantation is a recognized curative treatment for WAS, but when a matched donor is not available, administration of WAS gene-corrected autologous HSCs represents a valid alternative therapeutic approach. Since alterations of WAS protein (WASp)-deficient B lymphocytes contribute to immunodeficiency and autoimmunity in WAS, we followed the B cell reconstitution in 4 WAS patients treated by lentiviral vector-gene therapy (GT) after a reduced-intensity conditioning regimen combined with anti-CD20 administration. We analyzed the B cell subset distribution in the bone marrow and peripheral blood by flow cytometry and the autoantibody profile by a high-throughput autoantigen microarray platform before and after GT. Lentiviral vector-transduced progenitor cells were able to repopulate the B cell compartment with a normal distribution of transitional, naive and memory B cells. The reduction in the proportion of autoimmune-associated CD21low B cells and in the plasma levels of B cell-activating factor was associated with the decreased autoantibody production in WAS patients after GT. Then, we evaluated the functionality of B cell tolerance checkpoints by testing the reactivity of recombinant antibodies isolated from single B cells. Before GT, we found a decreased frequency of autoreactive new emigrant/transitional B cells in WAS patients, suggesting a hyperfunctional central B cell checkpoint in the absence of WASp. In contrast, high frequency of polyreactive and Hep2 reactive clones were found in mature naive B cells of WAS patients, indicating a defective peripheral B cell checkpoint. Both central and peripheral B cell tolerance checkpoints were restored after GT, further supporting the qualitative efficacy of this treatment. In conclusion, WASp plays an important role in the regulation of B cell homeostasis and in the establishment of B cell tolerance in humans and lentiviral-mediated GT is able to ameliorate the functionality of B cell compartment contributing to the clinical and immunological improvement in WAS patients.

Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Transcript expression of the freeze responsive gene fr10 in Rana sylvatica during freezing, anoxia, dehydration, and development.

Freeze tolerance is a critical winter survival strategy for the wood frog, Rana sylvatica. In response to freezing, a number of genes are upregulated to facilitate the survival response. This includes fr10, a novel freeze-responsive gene first identified in R. sylvatica. This study analyzes the transcriptional expression of fr10 in seven tissues in response to freezing, anoxia, and dehydration stress, and throughout the Gosner stages of tadpole development. Transcription of fr10 increased overall in response to 24 h of freezing, with significant increases in expression detected in testes, heart, brain, and lung when compared to control tissues. When exposed to anoxia; heart, lung, and kidney tissues experienced a significant increase, while the transcription of fr10 in response to 40% dehydration was found to significantly increase in both heart and brain tissues. An analysis of the transcription of fr10 throughout the development of the wood frog showed a relatively constant expression; with slightly lower transcription levels observed in two of the seven Gosner stages. Based on these results, it is predicted that fr10 has multiple roles depending on the needs and stresses experienced by the wood frog. It has conclusively been shown to act as a cryoprotectant, with possible additional roles in anoxia, dehydration, and development. In the future, it is hoped that further knowledge of the mechanism of action of FR10 will allow for increased stress tolerance in human cells and tissues.

Antigen-conjugated human IgE induces antigen-specific T cell tolerance in a humanized mouse model.

Dendritic cells (DCs) play an important role in immune homeostasis through their ability to present Ags at steady state and mediate T cell tolerance. This characteristic renders DCs an attractive therapeutic target for the induction of tolerance against auto-antigens or allergens. Accordingly, Ag-conjugated DC-specific Abs have been proposed to be an excellent vehicle to deliver Ags to DCs for presentation and tolerance induction. However, this approach requires laborious reagent generation procedures and entails unpredictable side effects resulting from Ab-induced crosslinking of DC surface molecules. In this study, we examined whether IgE, a high-affinity, non-cross-linking natural ligand of FcεRI, could be used to target Ags to DCs and to induce Ag-specific T cell tolerance. We found that Ag-conjugated human IgE Fc domain (Fcε) effectively delivered Ags to DCs and enhanced Ag presentation by 1000- to 2500-fold in human FcεRIα-transgenic mice. Importantly, this presentation resulted in a systemic deletion of Ag-specific T cells and prevented these mice from developing delayed-type hypersensitivity, which is critically dependent on Ag-specific T cell immunity. Thus, targeting FcεRI on DCs via Ag-Fcε fusion protein may serve an alternative method to induce Ag-specific T cell tolerance in humans.

ATP‐binding cassette transporter A1 (ABCA1) promotes arsenic tolerance in human cells by reducing cellular arsenic accumulation

Arsenic is a toxic element widely distributed in nature, such as water and soil. To survive this metalloid in the environment, nearly all organisms develop strategies to tolerate arsenic toxicity to some degree. Some arsenic-resistance genes have been identified in bacteria and yeast, but for mammals, especially humans, these genes are largely unknown. The aim of the present study was to identify these genes and benefit our intervention of arsenic resistance. We first established a human arsenic-resistant ECV-304 (AsRE) cell line and then used suppression subtractive hybridization and microarray analysis to identify arsenic-resistant genes in these cells. Of the significantly upregulated genes, three ATP-binding cassette (ABC) subfamily members, namely ABCA1, ABCE1 and ABCF1, were chosen for further study with RNA interference and overexpression analyses. The 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to determine the cell survival rate and the IC50 , whereas atomic fluorescence spectrophotometry was used to determine intracellular arsenic levels. We found that among the three ABC genes, only when ABCA1 gene expression was silenced did cells obviously lose their arsenic tolerance. The arsenic accumulation in ABCA1 deficiency AsRE cells was greater than that in wild type AsRE cells. Overexpression of ABCA1 in HeLa cells decreased arsenic accumulation in the cells and the cells were more resistant to As(III) than control cells transfected with empty vector. These results suggest a new functional role for ABCA1 in the development of arsenic resistance in human cells.

The establishment of early B cell tolerance in humans: lessons from primary immunodeficiency diseases

Patients with primary immunodeficiency (PID) provide rare opportunities to study the impact of specific gene mutations on the regulation of human B cell tolerance. Alterations in B cell receptor and Toll-like receptor signaling pathways result in a defective central checkpoint and a failure to counterselect developing autoreactive B cells in the bone marrow. In contrast, CD40L- and MHC class II-deficient patients only displayed peripheral B cell tolerance defects, suggesting that decreased numbers of regulatory T cells and increased concentration of B cell activating factor (BAFF) may interfere with the peripheral removal of autoreactive B cells. The pathways regulating B cell tolerance identified in PID patients are likely to be affected in patients with rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes who display defective central and peripheral B cell tolerance checkpoints. Indeed, risk alleles encoding variants altering BCR signaling, such as PTPN22 alleles associated with the development of these diseases, interfere with the removal of developing autoreactive B cells. Hence, insights into B cell selection from PID patients are highly relevant to the understanding of the etiology of autoimmune conditions.

Chronic Arsenic-Exposed Human Prostate Epithelial Cells Exhibit Stable Arsenic Tolerance: Mechanistic Implications of Altered Cellular Glutathione and Glutathione S-transferase

Acquisition of stable arsenic tolerance in human cells following chronic arsenic exposure has not been previously reported. In the present work, we describe acquisition of stable arsenic tolerance in the human prostate epithelial cell line RWPE-1 following chronic arsenic exposure in vitro. RWPE-1 cells continuously exposed to 5 μM sodium arsenite for ≥18 weeks exhibited dramatic resistance to acute arsenite toxicity. The LC50 for acute arsenite exposure in these chronic arsenic-exposed prostate epithelial (CAsE-PE) cells was 43.8 μM versus 17.6 μM in control cells. Similar results were obtained using the antineoplastic agent arsenic trioxide. This tolerance was stable, as CAsE-PE cells grown in arsenic-free medium for 5 weeks retained their resistant phenotype. Compared to control cells, CAsE-PE cells showed a 90% reduction in arsenic accumulation over 24 h coupled with a 2.6-fold increase in the rate of arsenic efflux. CAsE-PE cells had increased basal GSH levels (4.9-fold) and increased GST activity (2.4-fold) and both GSH depletion and inhibition of GST activity abolished arsenic tolerance. Arsenic tolerance was also abolished by treatment with inhibitors of the Mdr1 and Mrp1 transporters, although no increases in mdr1 or mrp1 gene expression were observed. Our results indicate that this tolerance in human cells involves increases in GSH levels and GST activity that allow for more efficient arsenic efflux by MRP1 and MDR1. This study represents the first report of stable acquired arsenic tolerance in human cells, which could have important implications for both the toxicology and the pharmacology of arsenic.

Chronic Arsenic-Exposed Human Prostate Epithelial Cells Exhibit Stable Arsenic Tolerance: Mechanistic Implications of Altered Cellular Glutathione and Glutathione S-transferase

Acquisition of stable arsenic tolerance in human cells following chronic arsenic exposure has not been previously reported. In the present work, we describe acquisition of stable arsenic tolerance in the human prostate epithelial cell line RWPE-1 following chronic arsenic exposure in vitro. RWPE-1 cells continuously exposed to 5 μM sodium arsenite for ≥18 weeks exhibited dramatic resistance to acute arsenite toxicity. The LC50 for acute arsenite exposure in these chronic arsenic-exposed prostate epithelial (CAsE-PE) cells was 43.8 μM versus 17.6 μM in control cells. Similar results were obtained using the antineoplastic agent arsenic trioxide. This tolerance was stable, as CAsE-PE cells grown in arsenic-free medium for 5 weeks retained their resistant phenotype. Compared to control cells, CAsE-PE cells showed a 90% reduction in arsenic accumulation over 24 h coupled with a 2.6-fold increase in the rate of arsenic efflux. CAsE-PE cells had increased basal GSH levels (4.9-fold) and increased GST activity (2.4-fold) and both GSH depletion and inhibition of GST activity abolished arsenic tolerance. Arsenic tolerance was also abolished by treatment with inhibitors of the Mdr1 and Mrp1 transporters, although no increases in mdr1 or mrp1 gene expression were observed. Our results indicate that this tolerance in human cells involves increases in GSH levels and GST activity that allow for more efficient arsenic efflux by MRP1 and MDR1. This study represents the first report of stable acquired arsenic tolerance in human cells, which could have important implications for both the toxicology and the pharmacology of arsenic.

Desiccation Tolerance in Human Cells

The ability to desiccate mammalian cells while maintaining a high degree of viability would have implications for many areas of biological science, including tissue engineering. Previously, we reported that introduction of the genes for trehalose biosynthesis allowed human cells in culture to be reversibly desiccated for up to 5 days. Here, we have further investigated the factors that allow human cells to survive in the desiccated state. The most important finding is that vacuum greatly enhances the ability of human cells in culture to withstand desiccation. In fact, cells dried slowly and stored under vacuum are able to withstand desiccation even in the absence of added carbohydrates or polyols. In addition to vacuum, the rate of desiccation, the temperature at which cells are maintained, the degree of confluence when dried, and the presence or absence of light have a large effect on the ability to retain viability in the desiccated state. Our data are consistent with a model in which cells can retain viability if they are desiccated in such a way that cellular structures are maintained. However, gradual loss of viability may be due to damage that occurs over time in the desiccated state, perhaps due to free radicals. Further optimization of the process for desiccating and maintaining cells is required before long-term storage of desiccated cells can be achieved.

Processing of psoralen adducts in an active human gene: Repair and replication of DNA containing monoadducts and interstrand cross-links

We have examined DNA repair in the dihydrofolate reductase (DHFR) gene in cultured human cells treated with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) using a newly developed assay for interstrand DNA cross-linking in defined genomic sequences. Within 24 hr, 80% of the cross-links, but only 45% of the monoadducts, were removed from a 32 kb transcribed sequence, demonstrating that repair efficiency in an active human gene varies with the nature of the damage. HMT monoadducts were also detected in the replicated DHFR sequence at frequencies indicating little interference with replication. The existence of cross-linkable monoadduct sites in the replicated DNA implies strand continuity opposite those sites and a relatively error-free mechanism of bypass. Translesion replication could circumvent transcription blockage in a damaged gene. These findings have important implications for mechanisms of mutagenesis and DNA lesion tolerance in human cells.