Reactive oxygen species (ROS) can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells. Previous in vitro biochemical assays have suggested a role for human Pol η in the insertion step of translesion synthesis (TLS) across the (5'S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol ι and yeast Pol ζ could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Pol η, Pol ι, and Pol ζ, but not Pol κ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol ι carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step.
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species generate many lesions in DNA, including R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG). Herein, the result of replication of a plasmid containing S-cdA in Escherichia coli is reported. S-cdA was found mutagenic and highly genotoxic. Viability and mutagenicity of the S-cdA construct were dependent on functional pol V, but mutational frequencies (MFs) and types varied in pol II- and pol IV-deficient strains relative to the wild type strain. Both S-cdA→T and S-cdA→G substitutions occurred in equal frequency in wild type E. coli, but the frequency of S-cdA→G dropped in pol IV-deficient strain, especially when SOS-induced. This suggests that pol IV plays a role in S-cdA→G mutations. MF increased significantly in pol II-deficient strain, suggesting pol II's likely role in error-free translesion synthesis. Primer extension and steady-state kinetic studies using pol IV, exo-free Klenow fragment (KF (exo-)), and Dpo4 were performed to further assess the replication efficiency and fidelity of S-cdA and S-cdG. Primer extension by pol IV mostly stopped before the lesion, although a small fraction was extended opposite the lesion. Kinetic studies showed that pol IV incorporated dCMP almost as efficiently as dTMP opposite S-cdA, whereas it incorporated the correct nucleotide dCMP opposite S-cdG 10-fold more efficiently than any other dNMP. Further extension of each lesion containing pair, however, was very inefficient. These results are consistent with the role of pol IV in S-cdA→G mutations in E. coli. KF (exo-) was also strongly blocked by both lesions, but it could slowly incorporate the correct nucleotide opposite them. In contrast, Dpo4 could extend a small fraction of the primer to a full-length product on both S-cdG and S-cdA templates. Dpo4 incorporated dTMP preferentially opposite S-cdA over the other dNMPs, but the discrimination was only 2- to 8-fold more proficient. Further extension of the S-cdA:T and S-cdA:C pair was not much different. For S-cdG, conversely, the wrong nucleotide, dTMP, was incorporated more efficiently than dCMP, although one-base extension of the S-cdG:T pair was less efficient than the S-cdG:C pair. S-cdG, therefore, has the propensity to cause G→A transition, as was reported to occur in E. coli. The results of this study are consistent with the strong replication blocking nature of S-cdA and S-cdG, and their ability to initiate error-prone synthesis by Y-family DNA polymerases.
Chemical Research in Toxicology 01/2014; 27(2). DOI:10.1021/tx4002786 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
Nature Chemical Biology 02/2014; DOI:10.1038/nchembio.1455 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the last three decades, a considerable amount of work has been undertaken to determine the nature, the mechanism of formation and the biological consequences of radiation-induced DNA lesions. Most of the information was obtained via the development of chemical approaches, including theoretical, analytical and organic synthesis methods. Since it is not possible to present all the results obtained in this review article, we will focus on recent data dealing with the formation of complex DNA lesions produced by a single oxidation event, as these lesions may play a significant role in cellular responses to ionizing radiation and also to other sources of oxidative stress. Through the description of specific results, the contribution of different chemical disciplines in the assessment of the structure, the identification of the mechanism of formation and the biological impacts in terms of repair and mutagenicity of these complex radiation-induced DNA lesions will be highlighted.
The British journal of radiology 03/2014; 87(1035):20130715. DOI:10.1259/bjr.20130715 · 2.03 Impact Factor
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