The excision of DNA lesions by human nucleotide excision repair (NER) has been extensively studied in human cell extracts. Employing DNA duplexes with fewer than 200 bp containing a single bulky, benzo[a]pyrene-derived guanine lesion (B[a]P-dG), the NER yields are typically on the order of ∼5–10%, or less. Remarkably, the NER yield is enhanced by a factor of ∼6 when the B[a]P-dG lesion is embedded in a covalently closed circular pUC19NN plasmid (contour length of 2686 bp) rather than in the same plasmid linearized by a restriction enzyme with the B[a]P-dG adduct positioned at the 945th nucleotide counted from the 5′-end of the linearized DNA molecules. Furthermore, the NER yield in the circular pUC19NN plasmid is ∼9 times greater than in a short 147-mer DNA duplex with the B[a]P-dG adduct positioned in the middle. Although the NER factors responsible for these differences were not explicitly identified here, we hypothesize that the initial DNA damage sensor XPC-RAD23B is a likely candidate; it is known to search for DNA lesions by a constrained one-dimensional search mechanism [Cheon, N. Y., et al. (2019) Nucleic Acids Res. 47, 8337–8347], and our results are consistent with the notion that it dissociates more readily from the blunt ends than from the inner regions of linear DNA duplexes, thus accounting for the remarkable enhancement in NER yields associated with the single B[a]P-dG adduct embedded in covalently closed circular plasmids.
The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1]>>[SpDNApl]) in contrast to monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with NEIL1-DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1-DNA complexes. The amplitude of the burst phase is limited in amplitude because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ » 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1-DNA complexes (τ-ns » 8 s). The estimated average DNA translocation distance of NEIL1 is ~80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of ~ 10.
The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) J. Biol. Chem. 321, 5309− 5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is ∼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by ∼20−30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.
As demonstrated by us earlier and by other researchers, a diet containing freeze-dried black raspberries (BRB) inhibits DNA damage and carcinogenesis in animal models. We tested the hypothesis that the inhibition of DNA damage by BRB is due, in part, to the enhancement of DNA repair capacity evaluated in the human HeLa cell extract system, an established in vitro system for the assessment of cellular DNA repair activity. The pre-treatment of intact HeLa cells with BRB extracts (BRBE) enhances the nucleotide excision repair (NER) of a bulky deoxyguanosine adduct derived from the polycyclic aromatic carcinogen benzo[a]pyrene (BP-dG) by ~24%. The NER activity of an oxidatively-derived non-bulky DNA lesion, guanidinohydantoin (Gh), is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Western Blot experiments indicate that the expression of selected, NER factors is also increased by BRBE treatment by ~73% (XPA), ~55% (XPB), while its effects on XPD was modest (<14%). These results demonstrate that BRBE significantly enhances the NER yields of a bulky and a non-bulky DNA lesion, and that this effect is correlated with an enhancement of expression of the critically important NER factor XPA and the helicase XPB, but not the helicase XPD.
We previously reported that black raspberries (BRB) powder inhibits dibenzo[def,p]chrysene (also known as dibenzo[a,l]pyrene; DBP)-induced DNA damage, mutagenesis and carcinogenesis in the mouse oral cavity. The effects of BRB on the capacity of DNA repair was investigated in an established in vitro system using HeLa cells to assess the DNA repair capacity. We found that pre-treatment of Hela cells with BRB extracts (BRBE) can enhance the nucleotide excision repair (NER) of a bulky DNA adduct derived from the tobacco oral carcinogen benzo[a]pyrene by ~24% and this effect is correlated with the enhanced expressions of NER factors XPA and XPB. The NER activity of guanidinohydantoin (Gh), an oxidatively-derived non-bulky DNA lesion, is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Using human oral leukoplakia (MSK-Leuk1) and oral squamous cell carcinoma SCC1483, we demonstrated that BRBE can inhibit DNA damage induced by DBP through the induction of GSH synthesis and enhancing GSH conjugates thereby inhibiting DNA damage. The effects of BRBE in MSK-Leuk1 and SCC1483 cells were further investigated on the DNA excision repair protein ERCC1 at 1, 6 and 24 h. ERCC1 forms the ERCC1-XPF enzyme complex that participates in several DNA repair pathways. Western blot analyses revealed that BRBE induced higher ERCC1 expression in SCC1483 than those observed in MSK-Leuk1 cells in a time-dependent manner. The results of our study demonstrate that BRBE is capable of inhibiting the metabolic activation, enhancing the detoxification pathway and DNA repair capacity which are consistent with its chemopreventive activity in vivo. Supported by NIH grant #CA173465. Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Ana H. Sales, Nicholas Geacintov, Karam El-Bayoumy. Effects of black raspberries extract on DNA repair capacity and metabolic activation/detoxification of the tobacco carcinogen dibenzo[def,p] in human oral cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4245.
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