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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2019)

Exposure to endogenous and exogenous factors could give rise to DNA damage. DNA alkylation constitutes a major form of DNA damage, and alkyl phosphotriesters (alkyl-PTEs) are particularly important owing to their relatively high frequencies of occurrence and resistance to repair in mammalian cells. However, little is known about how alkyl-PTEs influence fidelity and efficiency of DNA replication in cells and how translesion synthesis (TLS) polymerases modulate the replicative bypass of these lesions.

In Chapter 2. We synthesized and characterized oligodeoxynucleotides (ODNs) containing site-specifically inserted alkyl-PTEs (Me, Et, nPr and nBu) with different alkyl groups and diastereomeric configurations (Sp and Rp), and assessed how these lesions impact DNA replication in E. coli cells. Our results revealed that Sp-alkyl-PTEs could be efficiently bypassed, whereas their Rp counterparts exhibited moderate blockage effects on DNA replication. Additionally, Sp-Me-PTE was found to induce TTGT and TTGC mutations, which necessitated Ada proteins.

In Chapter 3. We prepared Me- or nBu-PTE-containing ODNs in two diastereomeric forms with different flanking dinucleotides sequences (XT and TX, X=A, C, G) and examined how these lesions were recognized by DNA replication machinery in E. coli cells. We found that Sp-XT-Me-PTEs were efficiently bypassed with similar distributions in replication products (with approximately 85-90% AT and 5-10% TG at XT site). where Ada protein is essential for the generation of the mutagenic products. Additionally, Rp-XT-Me-PTEs and both diastereomers of TX-Me-PTEs displayed error-free bypass with significant blockage effects. We also found that Ada binds strongly to Sp-Me-PTE at AT site, but not TA site. Our results suggest that the role of Ada may be beyond the repair of Sp-Me-PTE lesion and transcriptional modulation of genes involved in repairing alkylated DNA lesions.

In Chapter 4, we analyzed the cytotoxic and mutagenic properties of pyridyloxobutyl (POB)-PTEs in E. coli cells. We demonstrated that POB-PTEs did not display strong impediments to DNA replication, and replicative bypass of POB-PTEs does not require TLS polymerases. In Addition, POB-PTEs were not mutagenic.

Together, the research described in this dissertation constitutes the first chemical syntheses of ODNs harboring stereochemically defined alkyl-PTE lesions, and provides a much better understanding about the biological consequences of alkyl-PTE lesions.

    Cover page: Chemical Syntheses and Cellular Replication Studies of DNA Alkyl Phosphotriester Lesions
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2020)
    Genome integrity is constantly challenged by endogenous or exogenous genotoxic agents, which can give rise to various DNA adducts. After metabolic activation, tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can lead to pyridyloxobutylphosphotriesters (POB-PTEs) in DNA. Here, we synthesized oligodeoxyribonucleotides containing a site-specifically inserted SP- or RP-POB-PTE flanked by two thymidines, and we examined the impact that these lesions have on DNA replication in Escherichia coli cells. We found that these two lesions are not strong impediments to DNA replication, and their replicative bypass is not modulated by genetic depletion of the three SOS-induced DNA polymerases or Ada protein. In addition, neither SP- nor RP-POB-PTEs was mutagenic in E. coli cells. Together, our study unveiled, for the first time, the influence of tobacco-specific nitrosamine-induced POB-PTE lesions on DNA replication in vivo.
      Cover page: Replication of Pyridyloxobutyl Phosphotriester Lesions in Cells.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2018)
      Exposure to many endogenous and exogenous agents can give rise to DNA alkylation, which constitutes a major type of DNA damage. Among the DNA alkylation products, alkyl phosphotriesters have relatively high frequencies of occurrence and are resistant to repair in mammalian tissues. However, little is known about how these lesions affect the efficiency and fidelity of DNA replication in cells or how the replicative bypass of these lesions is modulated by translesion synthesis DNA polymerases. In this study, we synthesized oligodeoxyribonucleotides containing four pairs (Sp and Rp) of alkyl phosphotriester lesions at a defined site, and examined how these lesions are recognized by DNA replication machinery in Escherichia coli cells. We found that the Sp diastereomer of the alkyl phosphotriester lesions could be efficiently bypassed, whereas the Rp counterparts moderately blocked DNA replication. Moreover, the Sp-methyl phosphotriester induced TT→GT and TT→GC mutations at the flanking TT dinucleotide site, and the induction of these mutations required Ada protein, which is known to remove efficiently the methyl group from the Sp-methyl phosphotriester. Together, our study provided a comprehensive understanding about the recognition of alkyl phosphotriester lesions by DNA replication machinery in cells, and revealed for the first time the Ada-dependent induction of mutations at the Sp-methyl phosphotriester site.
        Cover page: Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2021)
        Alkylation represents a main form of DNA damage. The N2 position of guanine is frequently alkylated in DNA. The SOS-induced polymerases have been shown to be capable of bypassing various DNA damage products in Escherichia coli. Herein, we explored the influences of four N2-alkyl-dG lesions (alkyl = ethyl, n-butyl, isobutyl, or sec-butyl) on DNA replication in AB1157 E. coli cells and the corresponding strains with polymerases (Pol) II, IV, and V being individually or simultaneously knocked out. We found that N2-Et-dG is slightly less blocking to DNA replication than the N2-Bu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N2-alkyl-dG adducts, and none of these lesions was mutagenic. Together, our results support that the efficient replication across small N2-alkyl-dG DNA adducts in E. coli depends mainly on Pol II.
          Cover page: DNA Polymerase II Supports the Replicative Bypass of N2-Alkyl-2-deoxyguanosine Lesions in Escherichia coli Cells.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2020)
          Alkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- and n-butyl (nBu)-PTEs in two diastereomeric configurations (Sp and Rp) at six different flanking dinucleotide sites, i.e. XT and TX (X = A, C, or G), and assessed how these lesions impact DNA replication in Escherichia coli cells. When single-stranded vectors contained an Sp-Me-PTE in the sequence contexts of 5-AT-3, 5-CT-3, or 5-GT-3, DNA replication was highly efficient and the replication products for all three sequence contexts contained 85-90% AT and 5-10% TG. Thus, the replication outcome was largely independent of the identity of the 5 nucleotide adjacent to an Sp-Me-PTE. Furthermore, replication across these lesions was not dependent on the activities of DNA polymerases II, IV, or V; Ada, a protein involved in adaptive response and repair of Sp-Me-PTE in E. coli, however, was essential for the generation of the mutagenic products. Additionally, the Rp diastereomer of Me-PTEs at XT sites and both diastereomers of Me-PTEs at TX sites exhibited error-free replication bypass. Moreover, Sp-nBu-PTEs at XT sites did not strongly impede DNA replication, and other nBu-PTEs displayed moderate blockage effects, with none of them being mutagenic. Taken together, these findings provide in-depth understanding of how alkyl-PTE lesions are recognized by the DNA replication machinery in prokaryotic cells and reveal that Ada contributes to mutagenesis of Sp-Me-PTEs in E. coli.
            Cover page: Ada protein- and sequence context-dependent mutagenesis of alkyl phosphotriester lesions in Escherichia coli cells.
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2020)
            Chronic wounds are a major global health problem with the presence of biofilm significantly contributing to wound chronicity. Current treatments are ineffective in resolving biofilm and simultaneously killing the bacteria; therefore, effective biofilm-resolving drugs are needed. We have previously shown that, together with α-tocopherol, N-acetyl-cysteine (NAC) significantly improves the healing of biofilm-containing chronic wounds, in a diabetic mouse model we developed, by causing disappearance of the bacteria and breakdown of the extracellular polymeric substance (EPS). We hypothesize that NAC creates a microenvironment that affects bacterial survival and EPS integrity. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from diabetic mouse chronic wounds. For these studies, we chose mice in which chronic wound microbiome was rich in Pseudomonas aeruginosa (97%). We show that NAC at concentrations with pH < pKa causes bacterial cell death and breakdown of EPS. When used before biofilm is formed, NAC leads to bacterial cell death whereas treatment after the biofilm is established NAC causes biofilm dismantling accompanied by bacterial cell death. Mechanistically, we show that NAC can penetrate the bacterial membrane, increase oxidative stress, and halt protein synthesis. We also show that low pH is important for the actions of NAC and that bacterial death occurs independently of the presence of biofilm. In addition, we show that both the acetyl and carboxylic groups play key roles in NAC functions. The results presented here provide insight into the mechanisms by which NAC dismantles biofilm and how it could be used to treat chronic wounds after debridement (NAC applied at the start of culture) or without debridement (NAC applied when biofilm is already formed). This approach can be taken to develop biofilm from microbiome taken directly from human chronic wounds to test molecules that could be effective for the treatment of specific biofilm compositions.
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2022)
              Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.
                Cover page: Size- and Stereochemistry-Dependent Transcriptional Bypass of DNA Alkyl Phosphotriester Adducts in Mammalian Cells.
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2020)
                Chronic wounds are a major global health problem with the presence of biofilm significantly contributing to wound chronicity. Current treatments are ineffective in resolving biofilm and simultaneously killing the bacteria; therefore, effective biofilm-resolving drugs are needed. We have previously shown that, together with α-tocopherol, N-acetyl-cysteine (NAC) significantly improves the healing of biofilm-containing chronic wounds, in a diabetic mouse model we developed, by causing disappearance of the bacteria and breakdown of the extracellular polymeric substance (EPS). We hypothesize that NAC creates a microenvironment that affects bacterial survival and EPS integrity. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from diabetic mouse chronic wounds. For these studies, we chose mice in which chronic wound microbiome was rich in Pseudomonas aeruginosa (97%). We show that NAC at concentrations with pH < pKa causes bacterial cell death and breakdown of EPS. When used before biofilm is formed, NAC leads to bacterial cell death whereas treatment after the biofilm is established NAC causes biofilm dismantling accompanied by bacterial cell death. Mechanistically, we show that NAC can penetrate the bacterial membrane, increase oxidative stress, and halt protein synthesis. We also show that low pH is important for the actions of NAC and that bacterial death occurs independently of the presence of biofilm. In addition, we show that both the acetyl and carboxylic groups play key roles in NAC functions. The results presented here provide insight into the mechanisms by which NAC dismantles biofilm and how it could be used to treat chronic wounds after debridement (NAC applied at the start of culture) or without debridement (NAC applied when biofilm is already formed). This approach can be taken to develop biofilm from microbiome taken directly from human chronic wounds to test molecules that could be effective for the treatment of specific biofilm compositions.
                  Cover page: N‐Acetyl‐cysteine and Mechanisms Involved in Resolution of Chronic Wound Biofilm
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2017)
                  Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.
                    Cover page: Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis
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