The human DNA glycosylases NEIL1 and NEIL3 excise psoralen-induced DNA-DNA cross-links in a four-stranded DNA structure

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

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA–RPA and XPC–RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSβ were sensitive to psoralen ICLs. To further investigate the role of MutSβ in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSβ and NER proteins with Tdp-ICLs. We found that MutSβ bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSβ interacted with XPA–RPA or XPC–RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair

Etude de la voie de signalisation Sonic hedgehog dans les cancers cutanés de patients atteints de Xeroderma pigmentosum

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Human DNA polymerase iota protects cells against oxidative stress

Human DNA polymerase iota (polι) is a unique member of the Y-family of specialised polymerases that displays a 5′deoxyribose phosphate (dRP) lyase activity. Although polι is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biological importance of polι in human cells, we generated fibroblasts stably downregulating polι (MRC5-polιKD) and examined their response to several types of DNA-damaging agents. We show that cell lines downregulating polι exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H2O2) or menadione but not to ethylmethane sulphonate (EMS), UVC or UVA. Interestingly, extracts from cells downregulating polι show reduced base excision repair (BER) activity. In addition, polι binds to chromatin after treatment of cells with H2O2 and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged polι accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human polι in protecting cells from oxidative damage