The Human Oxidative DNA Glycosylase NEIL1 Excises Psoralen-induced Interstrand DNA Cross-links in a Three-stranded DNA Structure

Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1 excises photoactivated psoralen-induced monoadducts but not genuine interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the repair of ICLs in mammalian cells is mainly linked to DNA replication and proceeds via dual incisions in one DNA strand that bracket the cross-linked site. This process, known as “unhooking,” enables strand separation and translesion DNA synthesis through the gap, yielding a three-stranded DNA repair intermediate composed of a short unhooked oligomer covalently bound to the duplex. At present, the detailed molecular mechanism of ICL repair in mammalian cells remains unclear. Here, we constructed and characterized three-stranded DNA structures containing a single ICL as substrates for the base excision repair proteins. We show that NEIL1 excises with high efficiency the unhooked ICL fragment within a three-stranded DNA structure. Complete reconstitution of the repair of unhooked ICL shows that it can be processed in a short patch base excision repair pathway. The new substrate specificity of NEIL1 points to a preferential involvement in the replication-associated repair of ICLs. Based on these data, we propose a model for the mechanism of ICL repair in mammalian cells that implicates the DNA glycosylase activity of NEIL1 downstream of Xeroderma Pigmentosum group F/Excision Repair Cross-Complementing 1 endonuclease complex (XPF/ERCC1) and translesion DNA synthesis repair steps. Finally, our data demonstrate that Nei-like proteins from Escherichia coli to human cells can excise bulky unhooked psoralen-induced ICLs via hydrolysis of glycosidic bond between cross-linked base and deoxyribose sugar, thus providing an alternative heuristic solution for the removal of complex DNA lesions.Interstrand cross-links (ICLs)⁴ are highly lethal DNA lesions that block DNA transcription, replication, and recombination by preventing strand separation. Due to their high cytotoxicity, DNA cross-linking agents such as mitomycin C, cisplatin, and psoralens are widely used against hyperplasic diseases such as cancer and psoriasis (1, 2). Furanocoumarins (psoralens), naturally occurring secondary metabolites in plants, are tricyclic compounds formed by the fusion of a furan ring with a coumarin (Fig. 1). Among other ICL-inducing agents, psoralens require UVA photoactivation following DNA intercalation to chemically react with both cellular DNA in vivo and naked DNA in vitro (3). 8-Methoxypsoralen (8-MOP) is an asymmetric, planar compound that intercalates into DNA duplex near pyrimidines, preferentially at 5′-TpA sites. Upon photoactivation, 8-MOP primarily photoalkylates DNA by cycloaddition to the 5,6-double bond of a thymidine generating monoadducts (MA) with either the 4′,5′-double bond of the furan (MAf) or the 3,4-double bond of the pyrone (MAp) side of the psoralen (4) (supplemental Fig. S1). A unique property of psoralen photochemistry is that the absorption of a second photon by the MAf leads to formation of a pyrone side 5,6-double bond adduct with a flanking thymine in the complementary strand, thus generating an ICL (5). An angular fusion of the two-ring systems forms isopsoralens such as angelicin (Ang), 3-carbethoxypsoralen (3CP), and 7-methylpyrido(3,4-c)psoralen (MePy). In contrast to psoralens, isopsoralens, due to their geometry, cannot generate cross-links and form only monoadducts with DNA and RNA. The structures of several psoralen and isopsoralen derivatives are shown in Fig. 1.Open in a separate windowFIGURE 1.Chemical structure and numbering system for the furanocoumarins. A, 8-MOP; B, HMT; C, angelicin; D, 3CP; and E, MePy.The detailed structures of psoralen-DNA adducts including sites of covalent attachment of psoralen in DNA helix and chemical structure of psoralen-DNA adducts were well established (3). Crystal and NMR structures of psoralen-induced ICL revealed that psoralen can induce both minor and dramatic distortions into the DNA helix, depending on the sequence (6–8). This knowledge enabled geneticists to use photoactivated psoralen as a model agent to study ICL repair and mutagenesis. Importantly, up to 40% of induced DNA lesions by UVA+8-MOP exposure are ICLs, the remaining being two types of MAs (1). This is in striking contrast with other cross-linking agents such as mitomycin, cis-platin, and nitrogen mustard, which induce a plethora of different kinds of damage and only 1–8% of ICLs (1).Reconstitution of the repair of plasmids containing a single ICL in cell-free extract from Xenopus egg showed that ICL repair is coupled to DNA replication and involves convergence of two replication forks on the lesion (9). In mammalian cells, genetic evidence indicates that the repair of ICLs is also linked to DNA replication and proceeds via induction of a double strand break during DNA replication, probably due to replication fork collapse as a result of Mus81-Eme1 endonuclease cleavage of a template strand on the 3′-side of the lesion (10, 11). On the same strand, the XPF/ERCC1 nuclease might generate ICL-specific incision on the 5′-side of the damaged site to unhook a cross-linked DNA fragment from the template strand (12, 13). The resulting unhooked ICL swings free of the duplex, exposing a single-stranded gap. Translesion DNA synthesis (TLS) across the gap can be catalyzed by DNA polymerase κ (14) and/or by sequential action of Rev1 and polymerase ζ (15, 16), yielding a three-stranded DNA repair intermediate composed of a short oligomer covalently adducted to the duplex. This remaining cross-linked DNA fragment has to be removed from the template strand to unblock DNA replication. It has been proposed that in human cells, as in Escherichia coli and yeast, the unhooked ICLs are excised from the three-stranded DNA by the nucleotide excision repair (NER) machinery (17). However, with the exception of XPF/ERCC1-deficient cells, cells lacking critical NER components only show a moderate sensitivity to the cross-linking agents, which induce both MAs and ICLs (18, 19). These observations suggest that in human cells, either the NER pathway is only marginally responsible for the resolution of three-stranded DNA repair intermediates or an alternative pathway might exist.In the base excision repair (BER) pathway, a DNA glycosylase binds to the abnormal base by flipping it out of the helix and catalyzes cleavage of the base-sugar bond, generating either an abasic site or a single strand break with 3′-blocking moiety (20). Human homologues of the E. coli oxidized base-specific bifunctional DNA glycosylase/endonuclease VIII (Nei) were identified by a data base search of the genome and named NEIL1, -2, and -3 (human Nei-like proteins 1, 2, and 3) (21, 22). Recently, we demonstrated that the oxidative DNA glycosylases, E. coli Fpg and Nei and human NEIL1, excise with a high efficiency psoralen-induced MAs in duplex DNA. Consistent with this result, HeLa cells lacking APE1 and/or NEIL1 became hypersensitive to 8-MOP+UVA exposure (23), suggesting that BER may provide an alternative pathway to classic NER to eliminate bulky DNA adducts. The next step of our investigation was to examine how a genuine ICL in an oligonucleotide covalently linked to another oligonucleotide is repaired. For this purpose, we constructed a three-stranded DNA structure with unhooked ICL, a physiologically relevant structure derived from the endonuclease incisions of the template strand containing ICL, and examined its repair by the BER proteins. The present study provides new biochemical and genetic insights into the molecular mechanism of ICL repair.