DNA interstrand cross-links (ICLs) are among the most toxic lesions. They form impenetrable barrier for the DNA replication machinery and transcription. ICLs can be ge.nerated spontaneously or induced by environmental agents e.g. aldehydes or cisplatin. Different DNA repair pathway have been identified for repair of ICL [1]. One type of ICL is formed from an abasic site (Ap, from apurinic/apyrimidinc). Ap sites are very frequent DNA lesions arising spontaneously in genomic DNA by base loss. Two equilibrating forms of site cyclic hemiacetal and ring-opened aldehyde characterize Ap site and undergo amino-catalyzed strand cleavage generating an electrophilic 2,3-didehydro-2,3-dideoxyribose sugar remnant. A ring-opened aldehyde of Ap sites can react with primary amines of the residues on the opposing DNA strand [2]. ICL formed from the Ap site (Ap-ICL) is known to be repaired by Endonuclease VIII-like 3 (NEIL3). This repair has been demonstrated in DNA replication proficient Xenopus cell extracts and also in biochemical assays in vitro. During the replication fork cessation upon the Ap-ICL NEIL3 glycosylase is recruited to cleave the N-glycosidic bond unhooking the crosslink and allowing replication fork to proceed and extended the leading strand beyond the repaired lesion [3]. This work have revealed unprecedented repair machinery of Ap-ICLs. However, the mechanisms of crosslink recognition by the NEIL3 glycosylase remains unclear.
The focus of this work is the repair of spontaneously formed DNA crosslink formed within the DNA duplex. Our aim is determination of substrate specificity of NEIL3 glycosylase in order to understand the principles of substrate recognition and molecular mechanisms of the Ap-ICL repair. NEIL3 glycosylase contains three different domains. We have expressed and purified full-lenght and c-terminally truncated versions of NEIL3 glycosylase to reveal involvement of these domains in ICL repair. For enzymatic analysis we have prepared Ap-ICL in form of the DNA duplex and also different structures mimicking replication fork. We characterize structural and mechanistic features essential for the recruitment and Ap-ICL repair by NEIL3.
The project was supported by Czech Science Foundation: 17-21649Y and Academy of Sciences Czech Republic RVO: 61388963