Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes.

Human nucleotide excision repair processes carcinogen-DNA adducts at highly variable rates, even at adjacent sites along individual genes. Here, we identify conformational determinants of fast or slow repair by testing excision of N2-guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G x C-->T x A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (+)-trans-BPDE-N2-dG adduct 15 times less efficiently than a standard acetylaminofluorene-C8-dG lesion in the same sequence. No difference was observed between (+)-trans- and (-)-trans-BPDE-N2-dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (+)-cis- or (-)-cis-BPDE-N2-dG. Conversely, excision of (+)-cis- and (-)-cis- but not (+)-trans-BPDE-N2-dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N2-dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots.

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.

Impairment of nucleotide excision repair by apoptosis in UV-irradiated mouse cells.

We investigated the relationship between nucleotide excision repair (NER) activity and apoptosis in UV-irradiated cells. Mouse erythroleukemia (MEL) and lymphoma (GRSL) cells exhibited enhanced sensitivity to the cytotoxic effects of UV radiation compared to hamster cell lines, although normal UV-induced hprt mutation frequencies were found. Determination of UV-induced repair replication revealed a limited capacity of MEL and GRSL cells to perform NER consistent with poor removal of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts from transcriptionally active genes during the first 8 h after UV exposure. However, both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts appeared to be processed to almost normal level 24 h after UV treatment. In parallel, we observed that the UV-irradiated MEL and GRSL cells suffered from severe DNA fragmentation particularly 24 h after UV exposure. Taken together, these data indicate a reduced repair of UV-induced photolesions in apoptotic cells, already established at the early onset of apoptosis. To test whether inhibition of repair in cells was due to inactivation of NER or to apoptosis-induced chromatin degradation, we performed in vitro excision assays using extracts from UV-irradiated MEL cells. These experiments showed that the NER capacity during early apoptosis was intact, indicating that slow removal of UV-induced photolesions in apoptotic cells is due to substrate modification (presumably degradation of chromatin) rather than direct inhibition of factors involved in NER.

Site-specific DNA substrates for human excision repair: comparison between deoxyribose and base adducts.

BACKGROUND: The genetic integrity of living organisms is maintained by a complex network of DNA repair pathways. Nucleotide excision repair (NER) is a versatile process that excises bulky base modifications from DNA. To study the substrate range of this system, we constructed bulky deoxyribose adducts that do not affect the chemistry of the corresponding bases. These novel adducts were incorporated into double-stranded DNA in a site-specific manner and the repair of the modified sites was investigated. RESULTS: Using restriction enzymes as a probe for DNA modification, we confirmed that the resulting substrates contained the bulky deoxyribose adducts at the expected position. DNA containing these unique adducts did not stimulate DNA repair synthesis when mixed with an NER-competent human cell extract. Inefficient repair of deoxyribose adducts was confirmed by monitoring the release of single-stranded oligonucleotides during the excision reaction that precedes DNA repair synthesis. As a control, the same human cell extract was able to process a base adduct of comparable size. CONCLUSIONS: Our results indicate that modification of DNA bases rather than disruption of the sugar-phosphate backbone is an important determinant for damage recognition by the human NER system. Specific positions in DNA may thus be modified without eliciting NER responses. This observation suggests new strategies for anticancer drug design to generate DNA modifications that are refractory to repair processes.

Recognition of DNA adducts by human nucleotide excision repair. Evidence for a thermodynamic probing mechanism.

The mechanism by which mammalian nucleotide excision repair (NER) detects a wide range of base lesions is poorly understood. Here, we tested the ability of human NER to recognize bulky modifications that either destabilize the DNA double helix (acetylaminofluorene (AAF) and benzo[a]pyrene diol-epoxide (BPDE) adducts, UV radiation products) or induce opposite effects by stabilizing the double helix (8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 adducts). We constructed plasmid DNA carrying a defined number of each of these adducts and determined their potential to sequester NER factors contained in a human cell-free extract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. This novel approach showed differences of more than 3 orders of magnitude in the efficiency by which helix-destabilizing and helix-stabilizing adducts sequester NER factors. For example, AAF modifications were able to compete with the NER substrate approximately 1740 times more effectively than 8-MOP adducts. The sequestration potency decreased with the following order of adducts, AAF > UV >/= BPDE > 8-MOP > anthramycin, CC-1065. A strong preference for helix-destabilizing lesions was confirmed by monitoring the formation of NER patches at site-specific adducts with either AAF or CC-1065. This comparison based on factor sequestration and repair synthesis indicates that human NER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition involves thermodynamic probing of the duplex.

Stereoselectivity of human nucleotide excision repair promoted by defective hybridization.

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.

A repair competition assay to assess recognition by human nucleotide excision repair.

We developed a competition assay to compare, in a quantitative manner, the ability of human nucleotide excision repair (NER) to recognise structurally different forms of DNA damage. This assay uses a NER substrate consisting of M13 double-stranded DNA with a single and uniquely located acetylaminofluorene (AAF) adduct, and measures the efficiency by which multiply damaged plasmid DNA competes for excision repair of the site-directed modification. To validate this assay, we tested competitor DNA containing defined numbers of either AAF adducts or UV radiation products. In both cases, repair of the site-directed NER substrate was inhibited in a damage-specific and dose-dependent manner. We then exploited this competition assay to determine the susceptibility of bulky adozelesin-DNA adducts to human NER.

DNA synthesis arrest at C4'-modified deoxyribose residues.

Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives. A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases. Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack. We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct. Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested. These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties. However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates. The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.

Bipartite substrate discrimination by human nucleotide excision repair.

Mammalian nucleotide excision repair (NER) eliminates carcinogen-DNA adducts by double endonucleolytic cleavage and subsequent release of 24-32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose-phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4'-modified backbone residues. However, the same C4' backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose-phosphate composition. Thus, neither C4' backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4'-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson-Crick base pairing.

Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA.

Nondistorting C4' backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4' adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson-Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA-RPA complex may promote damage recognition by monitoring Watson-Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.