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Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.
Figure 1. Non denaturing PAGE analysis of the DNA structures containing single ICL used in DNA glycosylase activity assays. 5′-[32P]-labelled oligonucleotide strands are denoted by “*”, the presence of a crosslink is denoted by “-” and normal oligonucleotide base complementation is indicated by “∙”.
Figure 2. Action of Nei-like DNA glycosylases upon three-stranded DNA structure containing single HMT-derived ICL. (A) Denaturing PAGE analysis of the reaction products. 10 nM 5′-[32P]-labelled XL47∙47-21* was incubated for 1 hr at 37 °C either with the 500 nM NEIL3Cat, NEIL3Trun, NEIL3FL or with 50 nM NEIL1 proteins. Lanes 1–7, no piperidine treatment; lane 1, control non-treated XL47∙47-21*; lanes 2–4, as 1 but with NEIL3s; lane 5, NEIL1; lane 6, 21 mer duplex containing MA; lane 7, as 6 but Nei; lanes 8–11, as 1–4 but treated with light piperidine; lane 12, 21 mer D21; lanes 13–15, 21 mer U∙A duplex treated with UDG/hot piperidine, UDG/Nfo and UDG/light piperidine, respectively. Substrate and cleavage products sizes are indicated to the right of the gel. “Xâ€denotes substrate, “21-MA†denotes 21 mer fragment containing psoralen-derived MA, “21-mer†denotes size marker, “8 PAâ€, “8OH†and “8P†denote 8 mer fragments containing 3′-terminal PA, OH and P, respectively. For details see Materials and Methods. (B) Graphical representation of NEIL3s protein concentration dependent activities upon three-stranded DNA structure.
Figure 3. Schematic representation of the mechanisms of action of Nei-like DNA glycosylases on ICLs. (A) Mechanisms of action of Nei-like DNA glycosylases on three-stranded DNA structure containing single psoralen-derived ICL. For details see text. (B) Mechanisms of action of Nei-like DNA glycosylases on four-stranded DNA structure containing single psoralen-derived ICL. (C) Skeletal formula of 21 mer fragment containing single Thymidine nucleotide crosslinked to free Thymine base by HMT (DNA(T)-HMT-T), an excision product generated by Nei-like DNA glycosylases catalyzed repair of ICL in three-stranded DNA structure. (D) Skeletal formula of Thymine-HMT-Thymine cross-link (T-HMT-T), an excision product generated by Nei-like DNA glycosylases catalyzed repair of ICL in three- and four-stranded DNA structure.
Figure 4. Action of Nei-like DNA glycosylases upon four-stranded DNA structure containing single HMT-derived ICL. Denaturing PAGE analysis of the reaction products. 10 nM 5′-[32P]-labelled XL47∙47-21*∙21 was incubated for 1 hr at 37 °C either with the 500 nM NEIL3Cat, NEIL3Trun, NEIL3FL or with 20 nM Nei proteins. Lanes 1–7, no piperidine treatment; lane 1, control non-treated XL47∙47-21*∙21; lanes 2–4, as 1 but with NEIL3s; lane 5, as 1 but Nei, lane 6, 21 mer duplex containing MA; lane 7, as 6 but Nei; lanes 8–11, as 1–4 but treated with light piperidine; lane 12, 21 mer D21; lanes 13–15, 21 mer U∙A duplex treated with UDG/hot piperidine, UDG/Nfo and UDG/light piperidine, respectively. Substrate and cleavage products sizes are indicated to the right of the gel. “X†denotes substrate, “21-MA†denotes 21 mer fragment containing psoralen-derived MA, “21-mer†denotes size marker, “8 PAâ€, “8OH†and “8P†denote 8 mer fragments containing 3′-terminal PA, OH and P, respectively. For details see Materials and Methods.
Figure 5. Time dependent excision of three-stranded DNA structure containing single HMT-derived ICL by NEIL3Cat and NEIL1. (A) Denaturing PAGE analysis of the reaction products. 10 nM 5′-[32P]-labelled XL47∙47-21* was incubated with 300 nM NEIL3Cat and 50 nM NEIL1 at 37 °C for varied periods of time up to 1 h. Reaction products were not treated with piperidine. Lane 1, control non-treated XL47∙47-21*; lanes 2–7, as 1 but with NEIL3Cat; lanes 8–13, as 1 but with NEIL1. Substrate and cleavage products sizes are indicated to the right of the gel. “X†denotes substrate, “21-HMT-T†denotes 21 mer excision product containing HMT crosslinked free Thymine base, “21 mer†denotes 21 mer size marker. For details see Materials and Methods. (B) Graphical representation of NEIL3Cat and NEIL1 time kinetics on XL47∙47-21* DNA substrate. (C) Graphical representation of NEIL3Cat and NEIL1 time kinetics on 17 mer ssDNA and dsDNA fragment containing single Sp residue.
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