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Abstract
To assess helical parameters that dictate fast or slow removal of carcinogen-DNA adducts, we probed human nucleotide excision repair (NER) activity with DNA containing L-deoxyriboses. Unlike natural lesions such as pyrimidine dimers or base adducts, L-deoxyribonucleosides (the mirror images of normal D-deoxyribonucleosides) involve neither the addition nor the loss of covalent bonds or functional groups and hence exclude modulation of repair efficiency by adduct chemistry and size. Previous studies showed that single L-deoxyribonucleosides distort DNA backbones but are accommodated in the double helix with intact hydrogen bonding between complementary strands. Here, we found that such single L-enantiomers are rejected as excision repair substrates in a NER-proficient cell extract. However, the same L-deoxyribose moiety stimulates NER activity upon incorporation into a nonhybridizing site of one or, more effectively, two base mismatches. In contrast to single L-deoxyriboses, multiple consecutive L-deoxyriboses interfere with normal hybridization; in this case, the intrinsic derangement of base pairing was sufficient to promote the excision of a cluster of three adjacent L-deoxyribonucleosides without any requirement for mismatches. Thus, using stereoselective substrates, we demonstrate the participation of a recognition subunit that guides human NER activity to sites of defective Watson-Crick strand pairing. This conformational sensor detects labile hydrogen bonds irrespective of the type of deoxyribonucleotide modification.
Highlights
Mammalian nucleotide excision repair (NER)1 eliminates DNA damage with several overlapping levels of heterogeneity
Excision of Single L-Deoxyribose Substitutions—NER activity was determined using the oligonucleotide excision assay devised by Huang et al [21, 26]
We introduced a single L-deoxyribose at the thymine, guanine, or adenine position of the sequence 5Ј-TCGA-3Ј, but none of the tested deoxyribose replacements were able to elicit detectable NER activity in HeLa cell extract
Summary
Mammalian nucleotide excision repair (NER) eliminates DNA damage with several overlapping levels of heterogeneity. DNA repair is influenced by local chromatin compaction [14, 15]; at least in the human p53 gene, no clear correlation between the pattern of DNase I or micrococcal nuclease cleavage and the repair efficiency of UV-induced lesions could be detected [16] In view of these findings, we elaborated on the hypothesis that site-specific changes in DNA conformation may contribute significantly to the observed excision repair variability [17, 18]. The mirror image of natural D-deoxyribonucleosides was introduced into DNA to generate excision repair substrates without adding new chemical bonds (such as in pyrimidine dimers) or new functional groups (such as in carcinogen-DNA adducts) and without removing normal DNA components (such as in abasic sites, urea residues, formamidopyrimidines, or other base fragmentation products) This strategy was prompted by the expectation that the repairability of a model lesion that differs from normal DNA constituents only in its inverted stereochemistry might be susceptible to manipulation by altering the local double helical conformation. This study, which was conducted in the complete absence of base adducts or base degradation, indicates that repair heterogeneity is determined by the involvement of a NER subunit that senses improper base pairing at sites of DNA damage
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