Changjun You

A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses

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.

A quantitative assay for assessing the effects of DNA lesions on transcription

Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we described a novel competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrated that the oxidatively induced lesions 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG), and methylglyoxal-induced N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N2-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair (TC-NER) in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions impact DNA transcription in vitro and in cells.

The Roles of DNA Polymerases κ and ι in the Error-free Bypass of N2-Carboxyalkyl-2′-deoxyguanosine Lesions in Mammalian Cells

To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N2-modified 2′-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) and N2-carboxymethyl-2′-deoxyguanosine (N2-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N2-CEdG lesions. In contrast, no mutation was found after the N2-CEdG- and N2-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N2-CEdG and N2-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.

Endogenous formation and repair of oxidatively induced G[8-5 m]T intrastrand cross-link lesion

Exposure to reactive oxygen species (ROS) can give rise to the formation of various DNA damage products. Among them, d(G[8-5 m]T) can be induced in isolated DNA treated with Fenton reagents and in cultured human cells exposed to γ-rays, d(G[8-5m]T) can be recognized and incised by purified Escherichia coli UvrABC nuclease. However, it remains unexplored whether d(G[8-5 m]T) accumulates in mammalian tissues and whether it is a substrate for nucleotide excision repair (NER) in vivo. Here, we found that d(G[8-5 m]T) could be detected in DNA isolated from tissues of healthy humans and animals, and elevated endogenous ROS generation enhanced the accumulation of this lesion in tissues of a rat model of Wilson’s disease. Additionally, XPA-deficient human brain and mouse liver as well as various types of tissues of ERCC1-deficient mice contained higher levels of d(G[8-5 m]T) but not ROS-induced single-nucleobase lesions than the corresponding normal controls. Together, our studies established that d(G[8-5 m]T) can be induced endogenously in mammalian tissues and constitutes a substrate for NER in vivo.

Translesion Synthesis of 8,5'-Cyclopurine-2'-deoxynucleosides by DNA Polymerases η, ι, and ζ

Background: The 8,5′-cyclopurine-2′-deoxynucleosides (cPus) are important types of oxidative DNA damage. Results: cPus exhibit both inhibitory and mutagenic effects on replication; polymerases (Pols) η, ι, and ζ are involved in translesion synthesis of these lesions. Conclusion: Pols η, ι, and ζ cooperatively promote translesion synthesis across cPu lesions. Significance: This work revealed the effects of cPus on DNA replication in mammalian cells. Reactive oxygen species 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 polymerase (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 a two-subunit yeast Pol ζ complex (REV3/REV7) 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.

Identification of Novel α-N-Methylation of CENP-B That Regulates Its Binding to the Centromeric DNA

The eukaryotic centromere is an essential chromatin region required for accurate segregation of sister chromatids during cell division. Centromere protein B (CENP-B) is a highly conserved protein which can bind to the 17-bp CENP-B box on the centromeric DNA. In this study, we found that CENP-B could be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-Bs binding to CENP-B box in cells. Our study also expands the function of protein α-N-methylation that has been known for decades and whose function remains largely unexplored.

Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in vitro and in Human Cells

Background: Thiopurine prodrugs can result in the formation of S6-methylthioguanine (S6mG) and 6-thioguanine (SG) in DNA. Results: We examined how SG and S6mG affect DNA transcription in vitro and in human cells. Conclusion: S6mG, but not SG, causes a strong mutagenic and inhibitory effects on transcription. Significance: This work provides new knowledge that S6mG-mediated transcriptional alternations might contribute to thiopurine-induced cytoxicity and potential therapy-related cancers. Thiopurine drugs are extensively used as chemotherapeutic agents in clinical practice, even though there is concern about the risk of therapy-related cancers. It has been previously suggested that the cytotoxicity of thiopurine drugs involves their metabolic activation, the resultant generation of 6-thioguanine (SG) and S6-methylthioguanine (S6mG) in DNA, and the futile mismatch repair triggered by replication-induced SG:T and S6mG:T mispairs. Disruption of transcription is known to be one of the major consequences of DNA damage induced by many antiviral and antitumor agents; however, it remains undefined how SG and S6mG compromise the efficiency and fidelity of transcription. Using our recently developed competitive transcription and adduct bypass assay, herein we examined the impact of SG and S6mG on transcription in vitro and in human cells. Our results revealed that, when situated on the transcribed strand, S6mG exhibited both inhibitory and mutagenic effects during transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. Moreover, we found that the impact of S6mG on transcriptional efficiency and fidelity is modulated by the transcription-coupled nucleotide excision repair capacity. In contrast, SG did not considerably compromise the efficiency or fidelity of transcription, and it was a poor substrate for NER. We propose that S6mG might contribute, at least in part, to thiopurine-mediated cytotoxicity through inhibition of transcription and to potential therapy-related carcinogenesis via transcriptional mutagenesis.

Transcriptional inhibition and mutagenesis induced by N-nitroso compound-derived carboxymethylated thymidine adducts in DNA

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O4-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O4-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O4-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells.

Effects of Tet-mediated Oxidation Products of 5-Methylcytosine on DNA Transcription in vitro and in Mammalian Cells

5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

Aberrant transcription induced by DNA damage may confer risk for the development of cancer and other human diseases. Traditional methods for measuring lesion-induced transcriptional alterations often involve extensive colony screening and DNA sequencing procedures. Here we describe a protocol for the quantitative assessment of the effects of DNA lesions on the efficiency and fidelity of transcription in vitro and in mammalian cells. The method is also amenable to investigating the influence of specific DNA repair proteins on the biological response toward DNA damage during transcription by manipulating their gene expression. Specifically, we present detailed, step-by-step procedures, including DNA template preparation, in vitro and in vivo transcription, RNA purification, reverse-transcription PCR (RT-PCR) and restriction digestion of RT-PCR products. Analyses of restriction fragments of interest are performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and polyacrylamide gel electrophoresis (PAGE). The entire procedure described in this protocol can be completed in 15–20 d.