Remarkable Enhancement of Nucleotide Excision Repair of a Bulky Guanine Lesion in a Covalently Closed Circular DNA Plasmid Relative to the Same Linearized Plasmid

Abstract

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.

Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways

“…These lesions are substrates of prokaryotic BER and NER mechanisms [ 74 ], in cell extracts from HeLa cells and human fibroblasts [ 14 , 21 ], as well as in intact HeLa cells and human fibroblasts [ 19 ]. As a positive control of NER activity, we employed the bulky 10 R -(+)- cis-anti -B[ a ]PDE- N 2 -dG adduct (abbreviated as B[ a ]P-dG), an excellent substrate of the human NER pathway [ 14 , 19 , 22 , 32 , 33 ].…”

“…Recently we demonstrated that the interplay of BER and NER pathways in covalently closed circular plasmids dramatically differs from that in the same, but linearized plasmids [ 21 , 22 ]. A gapped-vector technology [ 75 , 76 , 77 , 78 , 79 , 80 ] was employed for the site-specific insertion of single guanine lesions into pUC19NN plasmid molecules (contour length of 2686 bp).…”

“…The formation of NER and BER excision products was monitored by 32 P-internally labelled plasmid substrates harboring single lesions, by high resolution denaturing polyacrylamide gel electrophoresis methods [ 21 , 22 ]. After incubation of the plasmid substrates in cell extracts, the DNA samples were isolated and treated with EcoRI and BsrBI restriction enzymes to excise 32 P-labeled 40-mer fragments, the products of successful BER activity, or the unincised and intact 101-mer fragments as shown in Figure 3 .…”

“…Successful NER activity generates the dual incision products ~24–32 nucleotides in lengths containing the 32 P-label, as well as the lesion [ 82 , 83 ]. A similar approach was employed to determine the yields of BER and NER excision products using linDNA containing single Sp and Gh lesions [ 21 , 22 ].…”

“…In this contribution, we summarize more recent experimental results that provide further insights into the competition between the BER and NER pathways in excising hydantoin lesions that are generated by the oxidation of guanine [ 14 , 18 , 19 , 20 , 21 ]. A significant enhancement (by factor of 5–6) of the NER dual incision yields of hydantoin lesions embedded in covalently closed circular DNA plasmids (cccDNA) than in the linearized form of the same plasmid DNA (linDNA) has been observed [ 21 , 22 ]. By contrast, the BER yields in cccDNA and linDNA did not vary by more than 20–40%, depending on the guanine lesion.…”