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Remarkable Enhancement of Nucleotide Excision Repair of a Bulky Guanine Lesion in a Covalently Closed Circular DNA Plasmid Relative to the Same Linearized Plasmid
journal contribution
posted on 2020-08-02, 14:29 authored by Marina Kolbanovskiy, Abraham Aharonoff, Ana Helena Sales, Nicholas E. Geacintov, Vladimir ShafirovichThe
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.